Virus-inspired compositions and methods of redirecting preexisting immune responses using the same for treatment of cancer

ABSTRACT

Disclosed are virus-inspired compositions and preparation methods thereof, where the compositions comprise mutant papillomavirus L1 proteins that spontaneously form capsid backbones and that are conjugated to a peptide comprising an epitope to form immune redirector capsids (IRCs). The epitopes on the peptides are designed to be recognized by a subject&#39;s immune system based on the subject&#39;s preexisting immune memory developed from the subject&#39;s past exposure to the epitope through infection or vaccination. The mutant papillomavirus L1 proteins possess three mutations including an amino-terminal truncation, a carboxy-terminal truncation, and a truncation at helix four. These mutations in the L1 protein yield capsomeres that are form non-canonical T=1 geometry capsid backbones. Disclosed are uses and methods of using the compositions in treating and/or preventing cancers in subjects in need thereof.

FIELD OF THE INVENTION

Disclosed are compositions comprising mutated papillomavirus proteins,especially the L1 major capsid protein, that form capsid backbones andare attached to one or more peptides comprising one or more antigensrecognized by a subject's preexisting immune system response memory, andtheir methods of use in treatment, prevention, and/or reduction in theincidence of cancer in a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationSer. No. 63/093,525, filed on Oct. 19, 2020, and U.S. provisional patentapplication Ser. No. 63/220,485, filed on Jul. 10, 2021, the entirecontents of each of which are hereby incorporated by reference.

INCORPORATION OF SEQUENCE LISTING

This application contains a sequence listing submitted in ComputerReadable Form (CRF). The paper copy of the sequence listing and the CRFare identical and are incorporated herein by reference. The sequencelisting contains one filed named “2021-10-19 8005US_ST25.txt” that wascreated on Oct. 19, 2021, and is 64 KB in size.

BACKGROUND

Typical cancer treatment includes chemotherapy, radiation, and surgery.However, surgery is highly invasive and often fails, especially aftermetastasis. Chemotherapy and radiation can be effective, but often yieldharsh side-effects that can drastically reduce quality of life forsubjects. Despite these treatments, many cancers remain refractory totreatment and the treatments can be ineffective in combating metastaticcancers even when successful in reducing or eliminating the primarytumor. Targeted delivery has become one of the most promisingopportunities for improving the treatment of cancer but this approachalso presents the most challenges. Immunotherapies such as cancervaccines have emerged as an attractive option due to the ability tostimulate the immune system and then use this response to specificallytarget over-expressed proteins preferentially present on the surface ofcancer cells, resulting in targeted elimination of the cancer cells.Such therapies are attractive in that they are target specific andpotentially less toxic without nonspecific autoimmunity. These targetedtherapies are also considered less invasive or traumatic compared tosurgery, radiation, or chemotherapy. However, cancer vaccines based oncancer-associated antigens can have limited success due to poor clinicalimmunogenicity, immune tolerance, and off target effects, for example.Moreover, such methods typically require identifying a cancer-associatedantigen specific to a given patient's cancer to achieve effectivetargeting of the cancer. Hence, this approach has failed on multipleoccasions because most cancer-associated antigens are self-antigens thatare tolerated by the immune system, resulting in poor immune responses.

Other approaches to the treatment and prevention of cancer are based onadoptive transfer of chimeric antigen receptor (CAR)-transduced T cells(CAR-T) or infusion of monoclonal antibodies that require the laboriousidentification of cancer-specific antigens and are applicable to only asubset of cancer types or subtypes. Finally, adoptive transfer oftumor-specific lymphocytes expanded ex vivo is a methodology that aimsto take advantage of naturally-occurring antitumor responses. All theseapproaches are similarly highly personalized and require theidentification cancer epitopes of the subject's specific cancer and/orexpansion of patient autologous cells ex vivo. Importantly, successesdemonstrated by these specific cancer antigen approaches in goldstandard animal models have not been always translatable to humans.Last, but not least, not all the patients suffering from cancer willexpress the same antigens on tumors, thus there are some significantlimitations to the broad applicability of these approaches.

A solution to the problem of individualized targeted treatment andelimination of cancer presents itself in the form of viral infectionhistory. In these approaches, a subject's infection history is used tore-initiate a past viral infection immune response through cytotoxicmemory T-cells. Such therapies based on past viral infections are finelytune-able to target specific cancers by depositing on the cancer cellsan epitope recognized by the subject's own immunological memory. VirusL1 proteins provide key functionality for delivering the epitope labelonto the cancer cell target, thereby recruiting and activating thesubject's own preexisting immune system components to target andeliminate the labeled cancer cells.

Mouse papillomavirus L1 proteins are good candidates for addressing thiscontinuing need for better, more personalized cancer treatments. It hasbeen fortuitously discovered that specific mutations in the mousepapillomavirus L1 protein lead to formation of smaller-sized T=1 viruscapsids, called capsid backbones, comprised of twelve (12) capsomeres,that are smaller than the normal T=7 capsids typically formed by virusL1 proteins, for instance as formed with human papillomavirus (HPV).These smaller-sized capsid backbones are very stable, allowing forhigher conjugation efficiency, and owing to their smaller size, presentless steric hindrance in infiltrating solid tumors or the tumormicroenvironment.

SUMMARY

In various embodiments, compositions comprising a plurality of mutantmouse papillomavirus L1 proteins are disclosed. The compositions furthercomprise one or more peptides that each comprise one or more epitopesfrom one or more pathogens other than a Papillomaviridae antigenicpeptide. The mutated amino acid sequence of the Papillomaviridae L1protein comprises at least the following mutations with respect to thewild type L1 protein sequence: (a) a deletion of at least five aminoacid residues from an amino-terminus, and (b) a deletion of at least tenamino acid residues from the helix four region. The one or more peptidesare attached to the plurality of virus proteins. The plurality of virusproteins spontaneously assemble to form an icosahedron or dodecahedroncapsid backbone having a triangulation number T equal to 1 that binds toproteoglycan expressed on tumor cells. Thus, the compositions comprise aplurality of mutant Papillomaviridae proteins and one or more suchpeptides. Said differently, the compositions comprise one or morepeptides attached to a plurality of mutant Papillomaviridae L1 proteins.

In some embodiments the mutant L1 proteins further comprise a deletionof at least thirty amino acid residues from the carboxy terminus of theL1 proteins. In some embodiments the peptides are conjugated to the L1proteins via disulphide, maleimide, or amide bond between the mutantPapillomaviridae L1 protein and a residue of the peptide.

In some embodiments from about 25% to about 85% (w/w) of the L1 proteinsare attached to at least one of the peptides. In some embodiments thepeptides also comprise a protease cleavage sequence, optionally selectedfrom a furin cleavage sequence, a matrix metalloprotease cleavagesequence, or a disintegrin and metalloprotease (ADAM) cleavage sequence.

The epitopes are not particularly limited other than that they should befrom an antigen that the subject to be treated has been previouslyexposed to and to which the subject has developed an immune reactivitytowards, or has an immune memory of the previous exposure such that uponre-exposure the subject's immune system will recognize and attack thecells bearing the epitopes. For instance, the epitope may be from achildhood vaccine. In other instances, the epitope may be from a pastpathogenic infection the subject recovered from.

The compositions comprising the Immune Redirector Capsid (IRC) moleculesbind to heparin sulfate proteoglycan located on cell surfaces. The IRCmolecules do not form T=7 capsids. In some embodiments, the mutant L1proteins are from mouse L1 proteins.

Contemplated herein are methods of treating, preventing, and/or reducingthe occurrence of cancer in a subject in need thereof, which comprisesadministering to the subject a pharmaceutically effective amount of thecompositions described herein. Also provided are methods of inhibitingcancer tumor growth, progression, and/or metastasis in a subject in needthereof, which comprises administering to the subject a pharmaceuticallyeffective amount of the compositions described herein. Uses of thedescribed compositions in the described methods are also contemplatedherein.

Such methods further comprise in some embodiments obtaining from thesubject a tumor tissue sample and identifying in the tumor tissue asequence of one or more MHC molecules expressed by one or more tumorcells in the tumor tissue sample.

In certain embodiments, the one or more epitopes are capable ofcomplexing with one or more MHC molecules expressed by a tumor cell in atumor tissue sample obtained from the subject. Secondary uses of thedescribed compositions are also contemplated, as in the use formanufacture of a medicament useful for such methods.

Further provided herein are processes for producing the describedcompositions. The processes include various steps, such as: (a)transforming a prokaryotic cell with an expression vector encoding theL1 protein's nuclei acid sequence; (b) culturing the transformedprokaryotic cell under conditions that promote expression of the L1protein; (c) lysing the transformed prokaryotic cells to releaseexpressed L1 protein; (d) separating cell debris from the expressed L1protein and recovering the L1 protein as inclusion bodies; (e)optionally washing the L1 protein inclusion bodies; (f) solubilizing theL1 protein inclusion bodies; (g) refolding the L1 protein in refoldingbuffer in the presence of reducing agent; and (h) forming theicosahedron or dodecahedron capsid having a triangulation number T equalto 1 in the same refolding buffer. Such processes, in some embodiments,further include conjugating in a conjugation buffer the one or morepeptides to the assembled L1 protein by incubating the assembled L1protein under reducing conditions in the presence of one or morepeptides and/or removing denaturant from the assembly buffer butmaintaining reducing agent when forming the icosahedron or dodecahedroncapsid having a triangulation number T equal to 1.

This Summary is neither intended nor should it be construed as beingrepresentative of the full extent and scope of the present disclosure.Moreover, references made herein to “the present disclosure,” or aspectsthereof, should be understood to mean certain embodiments of the presentdisclosure and should not necessarily be construed as limiting allembodiments to a particular description. The present disclosure is setforth in various levels of detail in this Summary as well as in theattached drawings and the Description of Embodiments and no limitationas to the scope of the present disclosure is intended by either theinclusion or non-inclusion of elements, components, etc. in thisSummary. Additional aspects of the present disclosure will becomereadily apparent from the Detailed Description, particularly when takentogether with the figures

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic gene map of wildtype mouse papillomavirus L1 (MPVL1) and associated regions.

FIG. 1B is a schematic gene map of triple truncated mouse papillomavirusL1 protein (MPV.10.34.d), multiple copies of which combine together toform the T=1 icosahedral papillomavirus capsid backbone.

FIG. 2 is an amino sequence alignment of MusPV L1 protein sequence (SEQID NO: 127) aligned with the corresponding human HPV16 L1 proteinsequence (SEQ ID NO: 128).

FIG. 3 is a flow chart of process steps leading to the manufacture ofMPV.10.34.d capsid backbones, which upon formation subsequently areconjugated to peptides to form immune regulatory capsids (IRCs).

FIG. 4A is a photograph of a stained SDS-PAGE gel showing theincremental purification of the MPV.10.34.d capsid backbone from hostcell protein contaminants through the manufacturing process (asdescribed in FIG. 3 and Example 1) demonstrating that host cell proteinimpurities are significantly reduced throughout the purification process(MW=standard molecular weight bands, numbers on the left-hand side ofthe gel indicate molecular weight in kiloDaltons (kDa), and top rownumbers indicate amount of protein loaded in micrograms).

FIG. 4B is a photograph of an SDS-PAGE gel subjected to silver staining,wherein each lane corresponds to a sample of MPV.10.34.d capsid backboneas it is purified from host cell protein contaminants through themanufacturing process (as described in FIG. 3 and Example 1),demonstrating that host cell protein impurities are significantlyreduced throughout the purification process (MW=standard molecularweight bands, numbers on the left-hand side of the gel indicatemolecular weight in kiloDaltons (kDa), and top row numbers indicateamount of protein loaded in micrograms).

FIG. 5 is a photograph of a transmission electron micrograph (TEM) imagewith a scale bar of 500 nm of the purified MPV.10.34.d capsid backbonefollowing the production and purification steps as described in FIG. 3and Example 1. The capsid backbone is approximately between 20 nm to 30nm in diameter based on this image.

FIG. 6A is a dynamic light scattering (DLS) plot showing the intensityof the particle size distribution of the MPV.10.34.d capsid backboneafter the refolding step but before the two-column chromatographypurification. The X-axis shows diameter size distribution (nm) and theY-axis provides percent intensity data.

FIG. 6B is a dynamic light scatting plot showing the population size ofpurified MPV.10.34.d capsid backbone after the refolding step but beforetwo-column chromatography purification. The X-axis shows diameter sizedistribution (nm) and the Y-axis provides volume percent data.

FIG. 7A is a dynamic light scattering plot showing MPV.10.34.d L1protein refolding in buffer with the following added: 1 mMdithiothreitol (DTT), 1 mM ethylenediamine tetraacetic acid (ETDA), and1 mM phenylmethylsulfonyl fluoride (PMSF). The X-axis shows diametersize distribution (nm) and the Y-axis provides volume percent data.

FIG. 7B is a dynamic light scattering plot showing MPV.10.34.d L1protein refolding in buffer with added 1 mM DTT. The X-axis showsdiameter size distribution (nm) and the Y-axis provides volume percentdata.

FIG. 7C is a dynamic light scattering plot showing MPV.10.34.d L1protein refolding in buffer with added 1 mM ETDA. The arrow indicatesaggregated protein that is not correctly refolded as detected by thevolume intensity plots, which measure population size of the sample as awhole. The X-axis shows diameter size distribution (nm) and the Y-axisprovides volume percent data.

FIG. 7D is a dynamic light scattering plot showing MPV.10.34.d L1protein refolding in buffer without any added DTT, EDTA, or PMSF. Thearrow indicates aggregated protein that is not correctly refolded asdetected by the volume intensity plots, which measure population size ofthe sample as a whole. The X-axis shows diameter size distribution (nm)and the Y-axis provides volume percent data.

FIG. 8A is a chromatogram showing capture of correctly refoldedMPV.10.34.d L1 protein refolded buffer with added 1 mM DTT, 1 mM ETDA,and 1 mM PMSF. The Y-axis provides absorbance units (mAU) on the leftand mS/cm on the right, with the X-axis indicating elution volume (mL).

FIG. 8B is a chromatogram showing capture of correctly refoldedMPV.10.34.d L1 protein refolded buffer with added 1 mM DTT. The Y-axisprovides absorbance units (mAU) on the left and mS/cm on the right, withthe X-axis indicating elution volume (mL).

FIG. 8C is a chromatogram showing capture of correctly refoldedMPV.10.34.d L1 protein refolded buffer with added 1 mM ETDA. The arrowindicates poor capture and elution, which indicates less correctlyrefolded MPV.10.34.d capsid backbone was observed under conditions C andD as compared with A and B. The Y-axis provides absorbance units (mAU)on the left and mS/cm on the right, with the X-axis indicating elutionvolume (mL).

FIG. 8D is a chromatogram showing capture of correctly refoldedMPV.10.34.d L1 refolded in the presence of just buffer with no addedDTT, EDTA, or PMSF. The arrow indicates poor capture and elution, whichindicates less correctly refolded MPV.10.34.d capsid backbone wasobserved under conditions C and D as compared with A and B. The Y-axisprovides absorbance units (mAU) on the left and mS/cm on the right, withthe X-axis indicating elution volume (mL).

FIG. 9A is a dynamic light scattering plot showing MPV.10.34.d L1protein showing particle size (diameter, nm) distribution (X-axis) basedon intensity (Y-axis) for eluted MPV.10.34d L1 protein, indicating anaverage particle size of 20 nm to 30 nm.

FIG. 9B is a dynamic light scattering plot showing MPV.10.34.d L1protein showing particle size (diameter, nm) distribution (X-axis) basedon volume (the relative proportion of refolded protein with respect toother contaminants, i.e., host bacterial cell proteins, Y-axis) foreluted MPV.10.34d L1 protein, indicating an average particle size of 20nm to 30 nm.

FIG. 9C is a photograph of a transmission electron microscopy (TEM)micrograph including a scale bar of 100 nm showing highly purifiedsoluble expressed MPV.10.34.d capsid backbone.

FIG. 10 is a graph of ELISA data obtained by binding a conformationalMPV L1-A4 conformation-specific monoclonal antibody to refoldedMPV.10.34.d capsid backbone showing that soluble and refoldedMPV.10.34.d capsid backbone have the same T=1 conformation underdifferent pH conditions. The Y-axis provides optical density at 450 nm,and the X-axis provides amount of MPV.10.34.d capsid backbone (ng).

FIG. 11 is a schematic representation of the steps to produce IRCs,i.e., conjugated MPV.10.34.d L1 capsid backbones.

FIG. 12 shows a photograph of a stained SDS-PAGE gel showingunconjugated MPV.10.34.d capsid backbones (lane 3) and IRCs (lane 4).Lane 1 shows molecular weight standards and lane 2 was intentionallyleft blank.

FIG. 13 is a photograph of a stained SDS-PAGE gel showing thatMPV.10.34.d capsid backbones experience a higher degree of peptideconjugation (lane 5) compared to wild type HPV16 capsid backbones (lane3). Lanes 2 and 4 are the unconjugated wild type HPV16 and MPV.10.34.dcapsid backbones, respectively. Lane 1 shows molecular weight standards.

FIG. 14 is a photograph of a stained SDS-PAGE gel showing samples ofwild type HPV16 IRC or MPV.10.34.d IRC or HPV16 capsid backbones in thepresence of reducing agent added in a ratio of reducing agent to L1protein of 0 (unconjugated controls, lanes 2 and 9), 10:1 (lanes 2 and10), 100:1 (lanes 3 and 11), and 1000:1 (lanes 4 and 12). The sampleswere then subjected to 10 rounds of filtration and then analyzed bySDS-PAGE in the same manner. Unconjugated controls are in lanes 5 and12, 10:1 ratio of reducing agent to L1 protein are in lanes 6 and 13,100:1 ratio of reducing agent to L1 protein are in lanes 7 and 14, and1000:1 ratio of reducing agent to L1 protein are in lanes 8 and 15.

FIG. 15A is a photograph of a TEM micrograph at 150,000× of wildtypeHPV16 IRC subjected to 10 rounds of filtration resulting in thebreakdown of the IRCs as seen in the circles.

FIG. 15B is a photograph of a TEM micrograph at 200,000× of wildtypeHPV16 IRC subjected to 10 rounds of filtration resulting in thebreakdown of the IRCs as seen in the circles.

FIG. 15C is a photograph of a TEM micrograph at 120,000× of MPV.10.34.dIRCs subjected to 10 rounds of filtration. The MPV.10.34.d IRCs did notdisplay any breakdown.

FIG. 15D is a photograph of a TEM micrograph at 200,000× of MPV.10.34.dIRCs subjected to 10 rounds of filtration. The MPV.10.34.d IRCs did notdisplay any breakdown.

FIG. 16 is a stained SDS-PAGE gel showing impact of peptideconcentration on peptide conjugation of MPV.10.34.d L1 peptidebackbones, where peptide is present during conjugation reactions inratios of 5:1, 10:1, and 25:1 of peptide:L1 when reducing agent((tris(2-carboxyethyl)phosphine, TCEP) is present at a ratio of 5:1reducing agent to L1 concentration (lanes 1, 4, and 7). No dependence ofconjugation rate on peptide concentration was seen in samples where thereducing agent to L1 ratio was 10:1 (lane 2, 5, and 8) or 20:1 (lanes 3,6, and 9). Lane 6 is a reference sample containing a ratio of 10:1reducing agent to L1 protein and 10:1 peptide to L1 concentration. (See,Example 6). This is a condition that has consistently providedapproximately 50% peptide conjugation as determined by densitometry. Thelane labelled CEX-FB035 (left side) is the MPV.10.34.d capsid backbonecontrol.

FIG. 17 is a stained SDS-PGE gel showing impact of peptide concentrationon peptide conjugation of MPV.10.34.d capsid backbones, where peptide ispresent during conjugation reactions in ratios of 5:1, 10:1, and 25:1 ofpeptide:L1 when reducing agent ((tris(2-carboxyethyl)phosphine, TCEP) ispresent at a ratio of 5:1 reducing agent to L1 concentration (lanes 3,6, and 9). Lanes 1, 4, and 7 are samples exposed to a ratio of 1:1reducing agent to L1 concentration. Lanes 2, 5, and 8 are samplesexposed to a ratio of 2.5:1 reducing agent to L1 concentration. R is areference sample containing a ratio of 10:1 of peptide to L1 and areducing agent to L1 ratio that has consistently provided approximately50% level of peptide conjugation as determined by densitometry. The lanelabelled CEX-FB035 (left side) is the MPV.10.34.d capsid backbonecontrol.

FIG. 18A is a flow cytometer histogram of cell count vs. fluorescenceintensity showing detection of the binding of MPV.10.34.d capsidbackbones and a variety of MPV.10.34.d IRCs showing that the IRCs retainspecificity to tumor cells via binding heparin sulfate proteoglycan(HSPG). Data include: MPV.10.34.d and corresponding CMV pp65 IRC (solidline), MPV.10.34.d and corresponding E7 IRC (thick solid line),MPV.10.34.d and corresponding OVA IRC (thick dashed line), andMPV.10.34.d capsid backbones (dashed-line). Samples exhibitedspecificity for tumor cells as evidenced by the shift of the peak to theright. The positive control in these experiments was wildtype MPV capsidbackbone (dotted line). The negative control included samples containingno IRC and no L1 (long-dashed line).

FIG. 18B is a flow cytometer histogram of cell count vs. fluorescenceintensity showing that all MPV.10.34.d IRCs do not bind to cells inwhich HSPG is not expressed. Data include: MPV.10.34.d and correspondingCMV pp65 IRC (solid line), MPV.10.34.d and corresponding E7 IRC (thicksolid line), MPV.10.34.d and corresponding OVA IRC (thick dashed line),and MPV.10.34.d capsid backbones (dashed-line). All samples exhibitedspecificity for tumor cells as evidenced by the shift of the peak to theright. The positive control in these experiments was wildtype MPV capsidbackbone (dotted line). The negative control included no IRC and no L1(long-dashed line).

FIG. 19 is a flow cytometer histogram of cell count vs. fluorescenceintensity showing detection of the tumor cell surface display of OVA(SIINFEKL, SEQ ID NO: 95)/Kb (MHC-I) complex. The results show OVA(SIINFEKL, SEQ ID NO: 95)-conjugated MPV.10.34.d IRCs are able to loadmore epitopes onto tumor cell MHC receptors as compared with OVA(SIINFEKL, SEQ ID NO: 95)-conjugated HPV16 IRCs, when equivalentmolarities of OVA-conjugated HPV16 IRCs (solid line) and OVA-conjugatedMPV.10.34.d IRCs (thick solid line) were compared side-by-side. Thenegative control is represented by a long-dashed line. The positivecontrol containing free peptide (SIINFEKL, SEQ ID NO: 95) at 1 μg/mL isrepresented by a short-dashed line.

FIG. 20A is a flow cytometer histogram of cell count vs. fluorescenceintensity showing detection of the tumor cell surface cell count of CMV(NLAPMVATV, SEQ ID NO: 129)/HLA-A*0201 (MHC-I) complex showingCMV-conjugated MPV.10.34.d IRCs are able to load human CMV viralepitopes onto human tumor cell MHC receptors in HCT116 cells. Datapoints include: unrelated control peptide (thin dashed line),MPV.10.34.d capsid backbone (thin solid line), MPV.10.34.d andcorresponding CMV pp65 IRC (thick solid line), and HCMV free peptide(thick dashed line).

FIG. 20B is a flow cytometer histogram of cell count vs. fluorescenceintensity showing detection of the tumor cell surface cell count of CMV(NLAPMVATV) (SEQ ID NO: 129)/HLA-A*0201 (MHC-I) complex.

FIG. 21A is a flow cytometer histogram of cell count vs. fluorescenceintensity showing detection of the showing competitive inhibition ofbinding of OVA-conjugated MPV.10.34.d IRCs to MC38 tumor cells with 10mg/mL soluble heparin (dashed line) pre-mixed into the sample. The solidline is a negative control showing no OVA peptide loading. The solidline that overlaps the dashed line is the negative control showing noOVA peptide loading.

FIG. 21B is a flow cytometer histogram of cell count vs. fluorescenceintensity showing detection of the competitive inhibition of binding ofOVA-conjugated MPV.10.34.d IRCs to MC38 tumor cells with 5 mg/mL solubleheparin (dashed line) pre-mixed into the sample. The solid line is anegative control showing no OVA peptide loading. The solid line thatoverlaps the dashed line is the negative control showing no OVA peptideloading. The solid line that overlaps the dashed line is the negativecontrol showing no OVA peptide loading.

FIG. 21C is a flow cytometer histogram of cell count vs. fluorescenceintensity showing detection of the competitive inhibition of binding ofOVA-conjugated MPV.10.34.d IRCs to MC38 tumor cells with 1 mg/mL solubleheparin (dashed line) pre-mixed into the sample. The solid line is anegative control showing no OVA peptide loading. The solid line thatoverlaps the dashed line is the negative control showing no OVA peptideloading.

FIG. 22A is a flow cytometer histogram of cell count vs. fluorescenceintensity showing that furin inhibitor at concentration of 50 μMprevents OVA-conjugated MPV.10.34.d IRCs from loading OVA epitopes ontotumor cell MHC receptors (arrow pointing to dark line). Samples includeuntreated cells (thin line) or cells treated only with dimethylsulfoxide (DMSO) and no furin inhibitor (dashed line), and negativecontrol (thin line).

FIG. 22B is a flow cytometer histogram of cell count vs. fluorescenceintensity showing that furin inhibitor at concentration of 5 μM preventsOVA-conjugated MPV.10.34.d IRCs from loading OVA epitopes onto tumorcell MHC receptors (arrow pointing to dark line). Samples includeuntreated cells (thin line) or cells treated only with dimethylsulfoxide (DMSO) and no furin inhibitor (dashed line), and negativecontrol (thin line).

FIG. 22C is a flow cytometer histogram of cell count vs. fluorescenceintensity showing that furin inhibitor at concentration of 0.5 μMprevents OVA-conjugated MPV.10.34.d IRCs from loading OVA epitopes ontotumor cell MHC receptors (arrow pointing to dark line). Samples includeuntreated cells (thin line), or cells treated only with dimethylsulfoxide (DMSO) and no furin inhibitor (dashed line), and negativecontrol (thin line).

FIG. 23A is a bar graph showing that OVA-conjugated MPV.10.34.d IRCselicit an immune redirection of OVA-specific murine T-cells similar toOVA-conjugated HPV16 IRC in murine tumor cell line ID8-Luc.

FIG. 23B is a bar graph showing that OVA-conjugated MPV.10.34.d IRCselicit an immune redirection of OVA-specific murine T-cells similar toOVA-conjugated HPV16 IRC in murine tumor cell lines B16-Luc.

FIG. 24A is a bar graph showing that E7-conjugated HPV16 IRCs elicit animmune redirection of OVA-specific murine T-cells similar toOVA-conjugated HPV16 IRC in murine tumor cell line ID8-Luc.

FIG. 24B is a bar graph showing that E7-conjugated HPV16 IRCs elicit animmune redirection of OVA-specific murine T-cells similar toOVA-conjugated HPV16 IRC in murine tumor cell lines B16-Luc.

FIG. 25A is a bar graph showing that human CMV-conjugated MPV.10.34.dIRCs elicit an immune redirection of CMV-specific CD8 T-cells similar toCMV-conjugated HPV16 IRCs in human tumor cell line HCT-116.

FIG. 25B is a bar graph showing that human CMV-conjugated MPV.10.34.dIRCs elicit an immune redirection of CMV-specific CD8 T-cells similar toCMV-conjugated HPV16 IRCs in human tumor cell line Ovarcar3.

FIG. 25C is a bar graph showing that human CMV-conjugated MPV.10.34.dIRCs elicit an immune redirection of CMV-specific CD8 T-cells similar toCMV-conjugated HPV16 IRCs in human tumor cell line MCF7.

FIG. 26 is a graph demonstrating high statistical correlation of bindingof OVA-conjugated MPV.10.34.d IRCs to tumor cells and peptide loading ofOVA (SIINFEKL, SEQ ID NO: 95) onto tumor cell surface MHC-I moleculesvia OVA-conjugated MPV.10.34.d IRCs.

FIG. 27A is a graph of percent cytotoxicity vs. E:T ratio showingblocking of binding of 0.625 μg/mL OVA-conjugated MPV.10.34.d IRCs byincubation with heparin at concentration of 10 mg/mL, an immuneredirection response of OVA-specific murine T-cells was not elicited(dashed line). The dotted line represents a negative control ofMPV.10.34.d capsid backbones. The solid line represents the positivecontrol sample with no heparin.

FIG. 27B is a graph of percent cytotoxicity vs. E:T ratio showingblocking of binding of 0.3125 μg/mL OVA-conjugated MPV.10.34.d IRCs byincubation with heparin at concentration of 10 mg/mL, an immuneredirection response of OVA-specific murine T-cells was not elicited(dashed line). The dotted line represents a negative control ofMPV.10.34.d capsid backbones. The solid line represents the positivecontrol sample with no heparin.

FIG. 27C is a graph of percent cytotoxicity vs. E:T ratio showingblocking of binding of 0.156 μg/mL OVA-conjugated MPV.10.34.d IRCs byincubation with heparin at concentration of 10 mg/mL, an immuneredirection response of OVA-specific murine T-cells was not elicited(dashed line). The dotted line represents a negative control ofMPV.10.34.d capsid backbones. The solid line represents the positivecontrol sample with no heparin.

FIG. 28 is a table of data obtained from a dose titration of thecell-binding assays and cytotoxicity assays. The study was repeatedtwice (with at least 3 replicates each). The mean values of geometricmean fluorescent intensity (MFI) are reported from the two experiments.These data were used to assess the statistical correlation ofOVA-conjugated MPV.10.34.d IRC binding and cytotoxicity.

FIG. 29 is a graph of percent cytotoxicity vs. MFI demonstrating highstatistical correlation of OVA-conjugated MPV.10.34.d IRC binding andcytotoxicity.

FIG. 30A is a graph of percent cytotoxicity vs. E:T ratio showing thatco-incubation of GARDASIL®9-vaccinated sera (1:200) with OVA-conjugatedMPV.10.34.d IRCs at 2.5 μg/mL did not inhibit immune redirection ofOVA-specific murine T-cells.

FIG. 30B is a graph of percent cytotoxicity vs. E:T ratio showing thatco-incubation of GARDASIL®9-vaccinated sera (1:200) with OVA-conjugatedMPV.10.34.d IRCs at 0.625 μg/mL did not inhibit immune redirection ofOVA-specific murine T-cells.

FIG. 30C is a graph of percent cytotoxicity vs. E:T ratio showing thatco-incubation of GARDASIL®9-vaccinated sera (1:200) with OVA-conjugatedMPV.10.34.d IRCs at 0.156 μg/mL did not inhibit immune redirection ofOVA-specific murine T-cells.

FIG. 31A is a graph of absorbance at 450 nm vs. dilution factor of dataobtained from two ELISA assays testing the effect of incubation ofanti-MPV.10.34.d sera with MPV.10.34.d on binding to target tumor cells.

FIG. 31B is a graph of absorbance at 450 nm vs. dilution factor of dataobtained from two ELISA assays testing the effect of incubation ofanti-MPV.10.34.d sera with MPV.10.34.d IRC. Results show that the seraspecifically recognize and bind to MPV.10.34.d capsid backbones.

FIG. 32A is a graph of percent cytotoxicity vs. E:T ratio showing thatco-incubation of MPV-specific sera (1:200) with OVA-conjugatedMPV.10.34.d IRC at 2.5 μg/mL did not inhibit immune redirection ofOVA-specific murine T-cells.

FIG. 32B is a graph of percent cytotoxicity vs. E:T ratio showing thatco-incubation of MPV-specific sera (1:200) with OVA-conjugatedMPV.10.34.d IRC at 0.625 μg/mL did not inhibit immune redirection ofOVA-specific murine T-cells.

FIG. 32C is a graph of percent cytotoxicity vs. E:T ratio showing thatco-incubation of MPV-specific sera (1:200) with OVA-conjugatedMPV.10.34.d IRC at 0.156 μg/mL did not inhibit immune redirection ofOVA-specific murine T-cells.

FIG. 32D is a graph of percent cytotoxicity vs. E:T ratio showing thatco-incubation of anti-MPV.10.34.d sera (1:200) with OVA-conjugatedMPV.10.34.d IRC at 2.5 μg/mL did not inhibit immune redirection ofOVA-specific murine T-cells, despite the fact that the sera specificallybound to OVA-conjugated MPV.10.34.d IRC.

FIG. 32E is a graph of percent cytotoxicity vs. E:T ratio showing thatco-incubation of anti-MPV.10.34.d sera (1:200) with OVA-conjugatedMPV.10.34.d IRC at 0.625 μg/mL did not inhibit immune redirection ofOVA-specific murine T-cells, despite the fact that the sera specificallybound to OVA-conjugated MPV.10.34.d IRC.

FIG. 32F is a graph of percent cytotoxicity vs. E:T ratio showing thatco-incubation of anti-MPV.10.34.d sera (1:200) with OVA-conjugatedMPV.10.34.d IRC at 0.156 μg/mL did not inhibit immune redirection ofOVA-specific murine T-cells, despite the fact that the sera specificallybound to OVA-conjugated MPV.10.34.d IRC.

FIG. 33A is a table of data corresponding to data graphed in FIGS. 32A,32B, and 32C, showing detection of the binding of OVA-conjugatedMPV.10.34.d IRC to tumor cells under different sample concentrations inthe presence of MPV serum (1:200) dilution. The results show binding ofOVA-conjugated MPV.10.34.d IRC despite the fact that MPV sera alsospecifically binds to OVA-conjugated MPV.10.34.d IRC.

FIG. 33B is a table of data corresponding to data graphed in FIGS. 32D,32E, and 32F, showing detection of the binding of OVA-conjugatedMPV.10.34.d IRC to tumor cells under different sample concentrations inthe presence of anti-MPV.10.34.d IRC serum (1:200) dilution. The resultsshow binding of OVA-conjugated MPV.10.34.d IRC to tumor cells despitethe fact that MPV.10.34.d IRC serum sera was shown to also specificallybind to OVA-conjugated MPV.10.34.d IRC.

FIG. 34 is a graph of geometric mean fluorescence intensity vs.concentration of OVA-conjugated MPV.10.34.d IRCs (ng/mL) detecting tumorcell surface display of OVA(SIINFEKL, SEQ ID NO: 95)/Kb (MHC-I) complex.The results show that OVA-conjugated MPV.10.34.d IRCs extra-cellularlyload OVA peptides onto MHC receptors on the surface of tumor cells thatare deficient in the MHC intracellular processing pathway.

DETAILED DESCRIPTION Definitions

This specification describes exemplary embodiments and applications ofthe disclosure. This disclosure, however, is not limited to theseexemplary embodiments and applications or to the manner in which theexemplary embodiments and applications operate or are described herein.Various embodiments, features, objects, and advantages of the presentteachings will be apparent from the description and accompanyingdrawings, and from the claims. As used herein, the terms “comprise,”“comprises,” “comprising,” “contain,” “contains,” “containing,” “have,”“having,” “include,” “includes,” and “including,” and their variants,are not intended to be limiting, are inclusive or open-ended, and do notexclude additional, unrecited additives, components, integers, elements,or method steps. For example, a process, method, system, composition,kit, or apparatus that comprises a list of features is not necessarilylimited only to those features but may include other features notexpressly listed or inherent to such process, method, system,composition, kit, or apparatus.

“About” is used to indicate that a value includes the standard deviationof error for the device or method being employed to determine the value.

“Immune redirector capsid” or “IRC” as used herein is a capsid backbonethat also comprises a peptide bound, attached, or conjugated, to thecapsid backbone.

“Cleavage sequence” as used herein includes, for example, specificpeptide sequences, or more often, peptide motifs at which site-specificproteases cleave or cut the protein. Cleavage sites are used, forexample, to cleave off an affinity tag, thereby restoring the naturalprotein sequence, or to inactivate a protein, or to activate proteins.In the present disclosure “cleavage” refers to proteolytic cleavage. Invarious embodiments, proteolytic cleavage is catalyzed by peptidases,proteases, or proteolytic cleavage enzymes before the final maturationof the protein. Proteins are also known to be cleaved as a result ofintracellular processing of, for example, misfolded proteins. Anotherexample of proteolytic processing of proteins is secretory proteins orproteins targeted to organelles, which have their signal peptide removedby specific signal peptidases before release to the extracellularenvironment or specific organelle. In one embodiment of the presentdisclosure, the cleavage sequence is specifically recognized by furinwhich cleaves and releases the peptides from the IRC, making the peptideavailable for loading onto or binding by the tumor cell surfacereceptors. In various embodiments, the cleavage sequence is comprised ofcysteine, lysine, and/or arginine residues, that not only allow thepeptide to be cleaved from the capsid backbone, but also serve asanchors to conjugate the peptide to the capsid protein until release bythe cleavage protein, such as furin, which are in some instancesenriched in, or selectively present at, the site of the tumor, i.e., inthe tumor microenvironment.

“Epitope” or “antigen” or “antigenic epitope” is a set of amino acidresidues that create recognition by or are recognized by a particularimmunoglobulin or, in the context of T cells, those residues necessaryfor recognition by T cell receptor proteins and/or majorhistocompatibility (MHC) receptors. The amino acid residues of anepitope need not be contiguous/consecutive. In an immune system setting,in vivo or in vitro, an epitope are in some instances a composite of thecollective features of a molecule, such as primary, secondary, andtertiary peptide structure, and charge, that together form athree-dimensional structure recognized by an immunoglobulin, T cellreceptor, and/or human leukocyte (HLA) molecule.

“HPV” and “human papillomavirus” refer to the members of the familyPapillomaviridae that are capable of infecting humans. There are twomajor groups of HPVs defined by their tropism (genital/mucosal andcutaneous groups), each of which contains multiple virus “types” or“strains/genotypes,” e.g., HPV 16, HPV 18, HPV 31, HPV 32, etc.

“MusPV,” “MMuPV1,” “MPV,” and “mouse papillomavirus,” all alternativelyand interchangeably refer to the known members of the familyPapillomaviridae that are capable of infecting mice (Mus musculus).

“Human vaccine” as used herein means a biological preparation thatimproves immunity to a particular disease in a human. A vaccinetypically contains an antigenic agent(s) that resembles adisease-causing agent (pathogen), and is often made from weakened orkilled forms of the microbe, its toxins, or one or multiple immunogenicsurface proteins of the disease-causing agent. The antigenic agentstimulates the body's immune system to recognize the disease-causingagent as foreign, destroy it, and “remember” it, so that the immunesystem can more easily recognize and destroy any of these pathogensshould an actual future infection/exposure occur. Human vaccines includevaccines against viral diseases and bacterial diseases. In variousembodiments, vaccines against viral diseases include hepatitis A, B, Evirus, human papillomavirus, influenza virus, Japanese encephalitisvirus, measles virus, mumps virus, polio virus, rabies virus, rotavirus,rubella virus, tick-borne encephalitis virus, varicella zoster virus,variola virus, and yellow fever virus. Human vaccines against viraldiseases that are under development include, for example, denguevaccine, eastern equine encephalitis virus, HTLV-1 T lymphocyte leukemiavaccine, and respiratory syncytial virus vaccine. Such a vaccineincludes, in some embodiments, current vaccines in development orcurrently United States Food and Drug Administration (FDA)-approvedvaccinations. A non-limiting list of examples of vaccines that arecompatible with the compositions and methods described herein isprovided in Table 2. The embodiments described herein, however, are notlimited to these listed vaccines, and are contemplated to apply to anyvaccine developed to provide immunity in a human subject.

“Inhibiting,” “reducing,” “prevention,” or “reducing the occurrence of,”and similar terms, when used herein, includes any measurable decrease orcomplete inhibition/reduction or elimination to achieve a desiredresult, such as inhibiting, reducing, or preventing, or reducing theoccurrence of, or reducing tumor mass, progression, and/or metastasis.

“MHC” or “major histocompatibility complex” is a group of genes thatencode proteins found on the surfaces of cells that help the immunesystem recognize foreign substances. MHC proteins (receptors, ormolecules) are expressed by all higher vertebrates. There are two maintypes of MHC molecules, MHC class I and MHC class II. In humans thereare three different genetic loci that encode MHC class I molecules (theMHC-molecules of the human are also designated human leukocyte antigens(HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A *02, and HLA-A*11 areexamples of different MHC class I alleles that can be expressed fromthese loci.

“Papillomavirus” (PV) refers to all members of the papillomavirus family(Papillomaviridae). An extensive list of papillomavirus types and theability to make the respective capsid backbones can be referenced usingthis publication: “Classification of papillomaviruses (PVs) based on 189PV types and proposal of taxonomic amendments,” de Villers et al.,401(1):70-79, 2010, PMID: 20206957 (all the tables specificallyincorporated herein by reference for all purposes).

“Preferentially cleaved protein” as used herein means that the peptideis preferentially cleaved from the capsid or capsomere or L1 protein atthe site of a tumor or tumor microenvironment. Without wishing to bebound by any particular theory, the preferential tumor-site cleavage isin some instances due to: (1) the unique cleavage sequence on thepeptide, and/or (2) the unique tumor microenvironment. For example, inone embodiment, the peptide comprises a cleavage sequence that ispreferentially cleaved by the enzyme furin, which is known to beexpressed in relatively higher concentrations around tumor cells ascompared with elsewhere in an organism.

“Protein,” “polypeptide,” and “peptide,” as used herein, are notrestricted to any particular number of amino acids; these terms aresometimes used interchangeably herein. The properties and amino acidsequences of the proteins described herein, and of the nucleic acidsencoding them, are well-known and are determined routinely, as well asdownloaded from various known databases. (See, e.g., the NCBI GenBankdatabases). Some peptide sequences are provided herein. However, somepeptide sequence information is routinely updated, e.g., to correctmistakes in the previous entries, so updated (corrected) informationabout the proteins and nucleic acids encoding them is included in thisapplication. Information provided in the sequence databases discussedherein is incorporated by reference.

An immune “response” is a humoral and/or cellular response of thesubject's immune system in which, in a cellular response, anantigen-primed cytotoxic T cell, Th1 T cell, Th2 T cell, and/or B cellsprimed by a vaccine or other pathogen present in the subject, or thatthe subject was previously exposed to, binds the epitope or antigen.

The term “preexisting immune response” as used herein means an immuneresponse that is present in an individual prior to initiation of theinventive cancer treatment methods described herein. Thus, an individualhaving a preexisting immune response has an immune response capacitystored within their memory T cells or other immune system componentsagainst an antigen, prior to the initiation of a method of treatment asdescribed herein with the antigen to treat cancer. A preexisting immuneresponse is in some instances a naturally-occurring immune response. Inother instances, the preexisting immune response is an induced immuneresponse. As used herein, a naturally-occurring preexisting immuneresponse is an immune response in an individual that was elicited inresponse to an antigen, such as a bacterial, fungal, parasitic, or viralantigen, with which the individual unintentionally contacted orcontracted. That is, an individual having a preexisting immune responsewas, in some instances, not exposed to an antigen with the intent togenerate an immune response to the antigen. An induced preexistingimmune response is an immune response resulting from an intentionalexposure to an antigen, such as when receiving a vaccine. Thepreexisting immune response is in some instances a naturally-occurringimmune response, or in other instances the preexisting immune responseis an induced immune response.

A “subject,” or “subject in need thereof,” as used herein, includes anyanimal that has a tumor/cancer or has had a tumor/cancer or has aprecancerous medical condition or cell or has a genetic or othersusceptibility, predisposition, or occupational risk of developingcancer or a tumor. Suitable subjects (patients) include laboratoryanimals, such as mouse, rat, rabbit, guinea pig, or pig, farm animals,such as cattle, sporting animals, such as dogs or horses, domesticatedanimals or pets, such as a horse, dog, or cat, nonhuman primates, andhumans.

“T cell response” as used herein refers to the immune response elicitedby T cells as they encounter antigens. Naïve mature T cells areactivated upon encountering antigen presented by B cells, macrophages,and dendritic cells, and then thereby produce armed effector T cells.Effector T cells are, in some instances, either CD8+ T cells thatdifferentiate into cytotoxic T cells, or CD4+ T cells that primarilyinduce the humoral immune response. The T cell immune response furthergenerates immunological memory that gives protection from the subsequentchallenge of the subject by the same or a similar pathogen comprisingthe same or similar epitopes. In various embodiments, the T cellresponse is at a threshold of at least 2-fold above the baseline oftotal CD8+ T cells. In various embodiments, the CD8+ T cells are CD69+as well.

“Therapeutic compositions” are compositions that are designed andadministered to patients for the use of treatment of a disease, such ascancer. Therapeutic compositions, e.g., therapeutic IRC-containingcompositions, are used to treat benign or malignant tumors orpatients/subjects at risk for such tumors, as well as non-solid cancers.In some embodiments, the IRCs are administered to a subject whopreviously had a tumor and is currently apparently tumor/cancer free, inan effort to enhance the inhibition or the recurrence of thetumor/cancer.

“Capsid backbone” refers to a multi-protein structure comprised of viralstructural proteins, such as envelop or capsid proteins, such as an L1protein, that in some instances self-assemble into a capsomere thatresembles a virus but lack viral genetic material. Capsid backbones arenon-infectious and non-replicating, yet morphologically similar toviruses. The capsid backbones disclosed herein bind to, or possess aninherent tropism for, tumor cells.

Capsid Structure

Viruses exist in many different morphologies and are generally smallerin size than bacteria, with a diameter between 20 nm and 300 nm,although some filoviruses possess filament lengths of up to 1400 nm.Visualization of viruses or virus capsid backbones requires transmissionelectron microscopes (TEM) that are more powerful than opticalmicroscopes. Viruses are particle in shape and exist as virions having anucleic acid surrounded by a protective coat of proteins called thecapsid. These capsids are also in turn in some instances surrounded by aprotective lipid bilayer that may include surface proteins, receptors,and the like.

Capsids are formed from a plurality of identical capsomeres. Capsidsgenerally fall into helical or icosahedral structures, with theexception of bacteriophages that possess more complex structures. Themost common icosahedral shape is composed of 20 equilateral triangularfaces and resembles a three-dimensional sphere in overall shape. Helicalcapsids resemble a common spring shape in the form of athree-dimensional cylinder. Each face of the capsid is comprised ofanywhere from one to three different proteins or monomer units(protomers). Capsids, when not surrounding papillomavirus genomes, arecommonly referred to in the art as virus-like particles, or hereinreferred to as capsid backbones. That is, an empty capsid with no viralgenomic material is referred to herein at times as a capsid backbone.Capsid backbones are excellent delivery molecules for treatment and/orprevention of various diseases, especially in the human body, becausethey are non-infectious and are optionally re-engineered to specificallytarget or bind to tumor cells, although most capsid backbone, asdescribed above, possess an inherent tissue tropism without furtherengineering.

Capsomeres are formed from individual subunits or protomers. Native L1protomers self-assemble through intermolecular disulfide bonds to formpentamers (capsomeres). As noted above, the capsid is comprised of manycapsomeres. As used herein, the term “capsomere” is intended to mean apentameric assembly of papillomavirus L1 polypeptides, includingfull-length L1 protein, or fragments and mutants thereof. A standardicosahedral capsid is comprised of twenty faces and is a polyhedronincluding twelve vertices. The vertices are comprised of pentagonalcapsomeres and the faces of the capsid are comprised of hexagonalcapsomeres. There are always twelve pentagons (pentons) and a varyingnumber of hexagons (hexons) in any given capsid depending on the virustype. Capsids that do not have an exogenous peptide attached thereto aretermed “capsid backbones” herein.

The icosahedral structure found in most viruses is very common andconsists of twenty triangular faces and twelve fivefold vertexes asnoted above. The number of capsomeres included in a capsid followswell-known mathematical principles, such as found in the Goldbergpolyhedron first described by Michael Goldberg in 1937. The structurescan be indexed by two integers h and k, with h being greater than orequal to one and k being greater than or equal to zero, the structure isvisualized by taking h steps from the edge of a pentamer, turning 60degrees counter-clockwise, then taking k steps to get to the nextpentamer. The triangulation number “T” for this type of capsid istherefore defined as T=h²+h·k+k². In this scheme, icosahedral capsidscontain twelve pentamers plus 10(T−1) hexamers. (See, Carrillo-Tripp, etal., Nuc. Acids Res., 37(Database issue):D436-D442, 2009). Thus, it canbe seen that the “T” number, or triangulation number, is representativeof the size and complexity of a given capsid. However, there are manyknown exceptions to this general “rule of thumb” found in, for instance,the Papillomaviridae family of viruses that can at times possesspentamers instead of hexamers in hexavalent positions, for instance in aquasi T=7 lattice. Outside of the canonical T=7 capsid structure, otherstructures such as T=1, T=2, and T=3, are known. A T=1 triangulationvalue indicates that the capsid is either only an icosahedron or adodecahedron.

Some viruses are enveloped and further comprise a lipid membrane coatingsurrounding the capsid structure. The envelope is acquired from the hostintracellular membrane. The nucleic acid material is either DNA or RNAand can be either single stranded or double stranded.

The Papillomaviridae family of viruses is a non-envelopeddouble-stranded DNA virus. There are several hundred family memberswithin the Papillomaviridae family, each of which is referred to as a“type” that infect most known mammals and other vertebrates such asbirds, snakes, turtles, and fish. The Papillomaviridae family membersare considered to be relatively highly host- and tissue-tropic, meaningthat its members usually possess a specific tissue tropism (preferencefor infection target) and a preference for host type, and are rarelytransmitted between species. For example, it is known that thePapillomaviridae family member human papillomavirus (HPV) type 1exhibits tropism for the soles of the feet, whereas HPV type 2 preferstissues in the palms of the hands. Papillomaviruses replicateexclusively in keratinocytes.

There are over 170 known human papillomavirus types that have beensequenced and are divided into five genera, including:Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus,Mupapillomavirus, and Nupapillomavirus. Many more human papillomaviruseshave been identified but not yet sequenced.

The papillomavirus has but a single protomer called L1 protein, or majorcapsid protein L1, that is both necessary and sufficient to form itscapsid which is comprised of 72 star-shaped capsomers. Thepapillomavirus family member capsids are non-enveloped and icosahedral.The papillomavirus genome also includes a second structural proteincalled L2 that is less abundantly expressed than L1. The presence of L2in the capsid is optional and not necessary for virus function or forformation of the capsid. All of the capsomeres of the Papillomaviridaefamily are made of pentamer interactions between proteins.

As described herein, when describing mutant L1 proteins and the like,such mutants, and capsomers, and capsids made therefrom, are meant toinclude all Papillomaviridae family members and not just human or mousefamily members. Thus, mutant L1 proteins as described herein are meantto encompass all L1 proteins in general, and in some instancesspecifically Papillomaviridae family L1 proteins in particular.

The amino acid domains and sequences of the human papillomavirus L1protein and its mouse counterpart are presented in FIGS. 1 and 2. Afairly high level of sequence conservation is generally observed acrossall such L1 proteins of the Papillomaviridae family and is alsoreflected in this alignment. Further shown in FIG. 2 are sites ofpossible mutation of the L1 sequence. Some of these mutations are knownhistorically, such as the deletion of ten amino acids from the amino- orN-terminus of the L1 protein. (See, for instance, Conway et al., J.Dent. Res., 88(4):307-317, 2009). Other structural mutations of thepeptide sequence of the L1 protein in Papillomaviridae family membersare known, such as the removal of the carboxy- or C-terminal residues ina truncation mutation.

The study of an N-terminal truncation mutant of L1 was begun partly inorder to obtain stable crystal structures of the protein for highresolution structural analysis of the capsid. Thus, it was found thatfull length HPV16 L1 were unable to be crystallized under most testedconditions, but upon removal of the ten N-terminal residues, a crystalwas able to be formed for further studies. (Conway et al., 2009).Surprisingly, it was found that upon removal of these ten N-terminalresidues, the capsomers formed a T=1 capsid structure comprisingicosahedral lattices made from twelve L1 pentamers (for a total of 60protomers). As noted above, the natural structure of thePapillomaviridae family member capsid is that of 72 L1 pentamers to forma T=7 structure. The T=1 structure of the N-terminal truncation mutantof HPV16 lacks certain disulfide bonds normally formed during capsidformation in wild type HPV16 capsids. Studies have shown that serine tocysteine mutation of C428 or deletion of the helix 4 region on humanpapillomavirus L1 capsid protein results in disrupting both the T=1 orthe T=7 capsid backbone formation. (See, Varsani et al., Virus Res.,122(1-2):154-163, 2006, and Schadlich et al., ibid.).

The overall structure of the papillomavirus L1 protein is presented inFIG. 1A and FIG. 1B and has a tertiary structure consisting generally ofvarious secondary structures including a core of β-strands that form aclassic “jelly roll” β-sandwich and five C-terminal α-helices thatsupport five surface-exposed loop regions generally designated in theart as loops BC, DE, EF, FG, and HI. (See, Chen et al., Mol. Cell,5:557-567, 2000, and Bissett et al., Scientific Reports, 6:39730, 2016).Three of the α-helices, commonly referred to as h2, h3, and h4, form thesurface of contact with other monomers and pentamers (FIG. 1B). (See,Chen et al., FIG. 4, page 561). The five α-helices generally reside atthe carboxy-terminus of the L1 sequence.

Design and Production of Mutant L1 Proteins

Deletions of the MPV L1 sequence were made to facilitating the formationof 10 nm to 15 nm capsomeres made from five L1 proteins. It waspreviously shown that truncation of the amino-, helix-four, andcarboxy-terminus residues of the HPV16 L1 protein results in capsomereformation. (See, Bishop et al., Virol. J., 4:3, 2007, and Schadlich etal., J. Virol., 83(15):7690-7705, 2009). On the other hand, it was shownin HPV11 and HPV16 that truncation of the amino-terminal ten residues ofL1, alone, would yield T=1 icosahedral capsid backbones. These T=1icosahedral capsid backbones are approximately 20 nm to 30 nm indiameter and consist of 60 L1 proteins (or 12 capsomers). Deletion of upto 34 amino acids at the carboxy-terminus did not inhibit T=1 formation.However, if deletions in the helix-four region of L1 occurred (aminoacids 411 to 436), the formation of T=1 would be ablated, even in thepresence of N-terminal or C-terminal truncations. In all permutations,capsomers would be observed. (Chen et al., Mol. Cell, 5(3):557-567,2000, and WO 2000054730). These results were consistent withpapillomavirus type 16 L1 produced in E. coli or in insect cells. (See,Schadlich et al., J. Virol., 83(15):7690-7705, 2009). Taken together,the authors of this study concluded that the helix-4 structure wasneeded for the assembly of capsomers into both higher ordered T=1 andT=7 icosahedral structures. (See, Bishop et al., Virol., 4:3, 2007).

Various deletions of the MPV L1 sequence were generated, resulting inthe construct called “MPV.10.34.d” shown schematically in FIG. 1B. Thisconstruct was designed to create an MPV capsomere as a therapeuticplatform. MPV L1 proteins were selected as the carrier vehicle constructinstead of HPV L1 because humans have for the most part not been exposedto MPV and therefore it was postulated that virion-derived capsids fromMPV would not be sensitive to innate immune response as is seen with HPVL1 proteins.

Recently a ΔN10 deletion of HPV16 L1, in which the amino-terminal tenresidues of the HPV L1 sequence are removed, was crystallized and foundto conform to the shape of a T=1 capsid backbone. (See, Chen et al.,2000). The structure revealed that the carboxy terminal segment fromresidue 384 to 446 of L1 folds into three helices with connecting loopsand turns. These helices are the primary inter-pentamer bonding contactsin the assembled T=1 capsid backbone. To test whether these helices alsoaffect capsid backbone assembly, L1 proteins comprising the ΔN10deletion were generated with a specific deletion of helix 4 for bothHPV16 (residues 408 to 431) and HPV11 (residues 409 to 429). Thepentamers were purified by FPLC and were shown to possess a “donut”shape as observed by electron microscopy (EM). No assembly of capsidbackbones from these pentamers was found under any condition tested,suggesting that this carboxy-terminal helical domain is essential forT=1 or T=7 capsid backbone assembly. Crystallographic analysis of theT=1 capsid backbone revealed that inter-pentameric contacts areestablished by hydrophobic interactions between the α-helices 2 and 3 ofone capsomere and α-helix 4 of a neighboring capsomere. (Chen et al.,2000). Consequently, a mutant L1 with helix 4 deleted formed homogenouscapsomeres but failed to form T=1 and T=7 capsid backbones. (See, Bishopet al., 2006). The constructs with helix 4 deleted did not exhibit anyability to self-assemble, consistent with previous reports. (See,Schadlich et al., J. Virol., 83(15):7690-7705, 2009).

For the purposes of this description, the term mutant L1 protein meansan L1 protein or protomer comprising one or more non-wild typesequences. Such non-wild type sequences include truncations or deletions(internal or at the ends of the sequences), single residuesubstitutions, and the like. For instance, a mutant L1 protein includesan L1 protein in which any of the following are true: 1) a certainnumber of the N-terminal residues are deleted, a certain number of theC-terminal residues are deleted, and/or 3) a certain number of internalresidues are deleted, in some instances in more than one locationinternally within the sequence.

The mutant L1 protein is in some embodiments derived from a wild typepapillomavirus L1 protein. Any papillomavirus L1 protein is useful inthe presently described compositions. L1 protein sequences arerelatively conserved. Thus, description of mouse papillomavirus mutantL1 proteins, below, are exemplary and it is contemplated that the samemutations made in other L1 proteins of the papillomavirus family isexpected to yield similar results. In various embodiments, a capsidbackbone is provided comprising a papillomavirus L1 protein and/or apapillomavirus L2 protein. Thus, the capsid backbone in some embodimentscomprises both papilloma L1 and L2 proteins. In other embodiments, thecapsid backbone is comprised of only L1 proteins. In some embodimentsthe L1 protein is a hybrid or chimeric protein comprised of L1 sequencesfrom more than one source merged together into a single L1 sequence.

The L1 protein sequences are known for substantially all papillomavirusgenotypes identified to date, and any of these L1 sequences or fragmentsare contemplated as being included in the present compositions. Examplesof L1 polypeptides include, without limitation, full-length L1polypeptides, e.g., HPV16 L1 polypeptide, SEQ ID NO: 128, L1 truncationsthat lack any one or more residues of the native C-terminus, L1truncations that lack any one or more residues of the native N-terminus,and L1 truncations that lack any one or more internal domain residues inany one or more internal locations. The L1 protein is in some instancesexemplified as a modified L1 protein, e.g., a modified HPV16 or MPV16 L1protein, wherein the HPV16 L2 amino acids 17 to 36 (the RG1 epitope) areinserted within the DE-surface loop of HPV16 L1. (See, Schellenbacher etal., J. Invest. Dermatol., 133(12):2706-2713, 2013; Slupetzky et al.,Vaccine, 25:2001-2010, 2007; Kondo et al., J. Med. Virol., 80:841-6,2008; Schellenbacher et al., J. Virol., 83:10085-10095, 2009; andCaldeira et al., Vaccine, 28:4384-93, 2010).

The L2 polypeptide is in some embodiments full-length L2 protein or anL2 polypeptide fragment. The L2 sequences are known for substantiallyall papillomavirus genotypes identified to date, and any of these L2sequences or fragments can be employed in the present disclosure.Examples of L2 polypeptides include, without limitation, full-length L2polypeptides, e.g., HPV16 L2 polypeptide (SEQ ID NO: 1), or mousepapillomavirus L2 (SEQ ID NO: 2), L2 truncations that lack any one ormore of the native C-terminus, L2 truncations that lack any one or moreof the native N-terminus, and L2 truncations that lack any one or moreinternal domain residues in any one or more locations.

The papillomavirus capsid backbone is in some embodiments formed usingthe L1 and optionally L2 polypeptides from any animal papillomavirus, orderivatives or fragments thereof. Thus, any known (or hereafteridentified) L1 and optionally L2 sequences of human, bovine, equine,ovine, porcine, deer, canine, feline, rodent, rabbit, etc.,papillomaviruses are employed to prepare the capsid backbones describedherein. (See, de Villiers et al., Virology, 324:17-27, 2004, for acurrent description of papillomavirus genotypes and their relatedness,incorporated herein by reference for all purposes).

In certain embodiments, the L1 and optionally L2 polypeptides that areused to form the capsid backbones are from a non-human papillomavirus ora human papillomavirus genotype other than HPV6, HPV11, HPV16, andHPV18. For example, the L1 and/or L2 proteins are in some embodimentsfrom HPV 1, 2, 3, 4, 5, 6, 8, 9, 15, 17, 23, 27, 31, 33, 35, 38, 39, 45,51, 52, 58, 66, 68, 70, 76, or 92.

As described above, in human papillomavirus HPV16, several differentmutations of L1 protein have been characterized. (See, for instance,Chen et al., 2000). Some of these mutations include the following inTable 1. (Chen et al., 2000, Table 1, page 558):

Trypsin Apparent Diameter of Deletion Sensitivity Assembled Particle(Å)^(a) ΔN = 0 No 600 ΔN = 8 No 600 ΔN = 9 No 600 ΔN = 10 No 300 ΔN = 15Yes^(b) NA ΔN = 20 Yes NA ΔC = 16 No 600 ΔC = 30 No 600 ΔC = 46 Yes NAΔC = 86 Yes NA

In Table 1, the delta symbol (A) designates deletion and the “N” or “C”designated whether the deletion is located at the N-terminus orC-terminus, respectively. The number following these two symbolsindicates the number of residues of the L1 sequence that were deleted.It is noted that Chen et al. does not report any double, triple, orhigher number of mutations within a single L1 protein.

Thus, the L1 mutant proteins described herein include N-terminaltruncation L1 mutant proteins. The N terminus is truncated by at least5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In someembodiments the N-terminal truncation is 5 amino acids. In someembodiments the N-truncation is 10 amino acids. In some embodiments theN-terminal truncation is 37, 38, 39, or even 40 amino acids.

The L1 mutant proteins described herein further include C-terminaltruncation L1 mutant proteins. The C terminus is truncated by at least5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In someembodiments the C-terminal truncation is 5 amino acids. In someembodiments the C-truncation is 10 amino acids.

The L1 mutant proteins described herein further include L1 mutants inwhich any number of internal residues are deleted. Surprisingly, theretention of the helix-4 region is in some embodiments needed for theformation of capsid backbones having a T=1 geometry, whereas in theliterature it is reported, as discussed above, that its deletion is notsupposed to yield any capsid backbone assembly. Generally, the internalresidues deleted in the described mutant L1 proteins are those shown inFIGS. 1 and 2. Contemplated also are deletions of 34 residues in thehelix 4 (H4) region. In some instances, the truncation of internalresidues of L1 proteins is 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, or even 40 residues in length.

Also described herein are L1 mutant proteins in which any one or more ofthe C-terminus and/or N-terminus and/or internal residues are deletedsimultaneously. For instance, in some embodiments the mutant L1 proteinhas both C- and N-terminal truncation mutations of similar or varyinglength. In other embodiments, the mutant L1 protein has a C-terminaltruncation and an internal residue truncation. In some embodiments, themutant L1 protein has an N-terminal truncation and an internaltruncation. In certain embodiments, the mutant L1 protein hastruncations simultaneously in all three locations, C-terminus,N-terminals, and internal truncations.

The mutant L1 proteins described herein are generally producedrecombinantly but are also produced by any known protein expressionmethodology. For instance, the mutant L1 proteins are generated by firstdesigning DNA primers complimentary to the wild type L1 sequence andthen performing PCR amplification of the sequence in the presence of theprimers designed to truncate or otherwise mutate the wild type L1sequence, as further explained in detail below, in the Examples section.(See also, Touze et al., J. Clin. Microbiol., 36(7):2046-2051, 1998).The design and implementation of proper primer sequences and PCRprotocols are known and such methods are used to ultimately generate thedesired mutant L1 protein nucleic acid sequence, from which the mutantL1 proteins are expressed.

The mutant L1 protein nucleic acid sequence is then in some embodimentscodon-optimized for better protein expression and production dependingon the organism in which the expression is conducted. Utilization ofdifferent codon optimization methods for certain expression vectors andhost expression systems are known in the art. (See, Mauro and Chappell,Trends in Molecular Medicine, 20(11):604-613, 2014, for instance).

The mutant L1 protein nucleic acid sequence is then ligated into anacceptably prepared and commercially-available expression vectordesigned for protein expression. Expression vectors of various typespossessing functionality for certain expression hosts are widelycommercially available. Recombinant mutant L1 proteins described hereinare expressed in bacterial as well as eukaryotic cells and in certainembodiments are expressible in vitro.

Often expression of recombinant proteins in bacterial hosts results inthe formation of inclusion bodies (IBs). Thus, in some instances,recombinant mutant L1 protein expressed as IBs are solubilized usingknown procedures. In a particular embodiment, the solubilization of IBsof expressed mutant L1 proteins described herein includes the steps setforth, for instance, in FIG. 3, and Example 3. The processes includevarious steps, such as: (a) transforming a prokaryotic cell with anexpression vector encoding the L1 protein; (b) culturing the transformedprokaryotic cell under conditions that promote expression of the L1protein; (c) lysing the transformed prokaryotic cells to releaseexpressed L1 protein; (d) separating cell debris from the expressed L1protein and recovering the L1 protein in the form of IBs; (e) optionallywashing the L1 protein IBs; (f) solubilizing the L1 protein IBs; (g)refolding the L1 protein optionally in the presence of one or moredenaturants, reducing agents, and the like; and (h) forming theicosahedron or dodecahedron capsid having a triangulation number T equalto 1 by incubating the refolded L1 protein in assembly buffer. Suchprocesses, in some embodiments, further include conjugating in aconjugation buffer the one or more peptides to the assembled L1 proteinby incubating the assembled L1 protein under reducing conditions in thepresence of one or more peptides and/or removing denaturant from theconjugation buffer but maintaining reducing agent when forming theicosahedron or dodecahedron capsid having a triangulation number T equalto 1.

The described methods and processes for creating and purifying thedescribed mutant L1 proteins is different in many aspects from suchprocesses described in the art for assembly of papillomavirus capsids.Indeed, it is known in the art that assembly into higher orderedpapillomavirus capsids requires that the L1 protein must first besubjected to a dis-assembly buffer that includes a reducing agent. Thisstep is then often followed by subjecting the L1 protein to an assemblybuffer that then removes the reducing agent. This legacy methodologyresults in stable capsids with improved properties. (See, McCarthy etal., 10.1128/JVI.72.1.32-41, 1998, Zhao et al., Virol. J., 9:52, 2012,Mach et al., J. Pharm. Sci., 95:2195-2206, 2006, and U.S. Pat. No.6,436,402).

Remarkable Properties of Capsid Backbones Formed From Mutant L1 Proteins

It was serendipitously discovered during the studies described hereinthat certain mutant L1 proteins possess beneficial and unexpectedproperties. For instance, certain mutant L1 proteins led primarily tothe formation of a T=1 capsid backbone possessing helpful and unexpectedconjugation properties. The formation of a T=1 capsid backbone insteadof a T=7 capsid backbone leads to higher stability under reducingconditions and therefore higher conjugation efficiency as compared withwild type sequences that form T=7 capsid backbone.

For instance, the efficiency with which the mutant capsid backbone,e.g., the MPV.10.34.d backbone, is able to be conjugated with peptide isfrom 25 to 85% (w/w). In some embodiments, the conjugation efficiency isabout 25%. In other embodiments, the conjugation efficiency is about 25,about 35, about 45, about 55, about 65, about 75, or even about 85%(w/w).

In contrast, wild type T=7 capsid backbones have generally a lowerefficiency of conjugation that is less than about 25%. See, forinstance, WO 2020/139978. The ability to achieve a higher amount ofpeptide conjugated to the T=1 capsid backbone compared to T=7 capsidbackbone allows for delivery of a higher number of peptides to thetarget tumor or cancer at an overall lower IRC dose amount compared withIRC forms from T=7 capsid backbones.

Additionally, T=1 capsid backbones having a smaller geometric shape orsize as compared to T=7 capsid backbones allows for less stearichindrance with the IRC made from T=1 capsid backbones is injected into asubject and the IRC infiltrate tumor microenvironments. This beneficialand unexpected effect then leads to a lower IRC dose needed to achievethe same effect as an equivalent T=7, or higher order, capsidbackbone-based IRC.

These and other additional beneficial features of the T=1 capsidbackbone geometry are described in further detail hereinbelow.

Mutant L1 Protein IRC and Mechanism of Action

In some embodiments, the mutant L1 protein is conjugated to anotherpeptide. To add further beneficial functionality to capsids or capsidbackbones comprised of the mutant L1 proteins, additional peptides areconjugated to the surface of such capsids. These peptides add beneficialfunctionality to the capsid and result in added functionality such astreatment of cancer in subjects in need thereof.

In an embodiment, the conjugated papillomavirus capsid backbonecomprises an L1 capsid protein and a peptide. In other embodiments, theIRC comprises an (at least one) L1 capsid protein, an (at least one) L2capsid protein, and at least one peptide. The L1 polypeptide is in someembodiments a full length L1 protein or in other embodiments is an L1polypeptide fragment. In specific embodiments, the full-length L1protein or L1 polypeptide fragment is capsid backboneassembly-competent; that is, the L1 polypeptide will self-assemble toform capsomeres under proper conditions that are competent forself-assembly into higher-order structural geometries, thereby forming acapsid backbone. In more specific embodiments, the capsid backbonescomprise a T=1 particle, a structure of about 20 nm to 30 nm indiameter, and composed of 12 capsomeres or 60 copies of L1 protein. Inother embodiments, the capsid backbones comprise a fully assembledpapillomavirus capsid, a structure of about 50 nm and composed of 72capsomeres or 360 copies of L1 protein.

In various embodiments, the IRC presented herein bind to, specificallyor non-specifically, or otherwise contact, one or more cancer cells.This is in part due to the capsid backbone's selectivity (tropism) forproteins and/or molecules that are in some instances specific to, orexpressed in higher abundance by, tumor cells. In various embodiments,the IRC binds to a certain sub-family type of heparin sulfateproteoglycan (HSPG), which is preferentially expressed on tumor cells.As used herein, “binding to a cancer cell” refers to the formation ofnon-covalent interactions between the capsid protein of the IRC and thetumor cell such that the IRC comes into close proximity to the tumorcell and the peptide is cleaved from the capsid backbone, and then thepeptide binds to, or is bound by, or otherwise interacts with, the MHCreceptor present on the tumor cell surface.

In various embodiments, the peptide is an epitope that is recognized bya T cell or T cell population that already exists in the subject. Invarious embodiments, this existing T cell or T cell population existsbecause of a prior infection or vaccination. In various embodiments, thepeptide is an epitope that is capable of being bound by a T cell. Invarious embodiments, the peptide is an epitope capable of being bound bya T cell already present in a subject. In this context, “capable ofbeing bound” means that an “epitope” is presented on the surface of acell, where it is bound to MHC molecules. T cell epitopes presentable byMHC class I receptors are bound by the T cell receptor of cytotoxic CD8T lymphocytes (CD8 T cells or CTLs). T cell epitopes presentable by MHCclass I molecules are typically peptides of about 9 to about 12 aminoacids in length. In various embodiments, an IRC is provided thatreleases a T cell response-eliciting peptide that upon release isdirectly bound by and consequently appropriately presentable by one ormore MHC molecules expressed on the surface of one or more cancer ortumor cells. As the released peptide does not require processing by theantigen processing machinery in the cytosol, the T cellresponse-eliciting peptides are presented on the surface of the targetcell in a short amount of time. The process of release of such peptidesfrom the IRC and subsequent binding of the peptides by the MHC moleculesof target cells is akin to labelling, tagging, or otherwise “marking”these tumor or cancer cells. This tagging or marking leads to readyidentification by other components of the subject's immune system,thereby recruiting these components of the subject's immune system toremove the cancer or tumor cells via the various known cell destructionpathways.

Hence, in one embodiment of the described methods, uses, andcompositions described herein, in less than about 8.5 hours afteradministration of the IRC dose to the subject, the IRC will naturallymigrate to the target cell after which the T cell response-elicitingpeptide released from the IRC, is bound by the MHC molecule on thecancer cell, and then the peptide is presented on the surface of thetarget cell via an MHC class I molecule to other components of thesubject's immune system for recognition thereby. In another embodimentof the invention, in less than 23.5 hours after introduction of the IRCto the target cell the T cell response eliciting peptide is presented onthe surface of the target cell via an MHC class I molecule. In anotherembodiment of the invention, the IRC is capable of mediating T cellcytotoxicity against the target cell within less than 6 hours afteradministration of the IRC to the target cell.

In various embodiments, the peptide comprises one epitope or comprisesat least two epitopes. The peptide epitopes are in some instancesderived from different proteins, or in other embodiments they areepitopes from the same protein (or antigen). In various embodiments, thepathogen is a virus, a bacterium, a fungus, a parasite, or a combinationthereof.

In various embodiments, the subject's preexisting T cells are specificto a vaccine epitope. In various embodiments the epitope is derived froma childhood, early childhood, adolescent, or elderly (geriatric),vaccine. In various embodiments the subject's preexisting immunity isthe result of prior administration of a human vaccine. Antigensdescribed herein that comprise epitopes incorporated into the peptidesdescribed herein are found in any of the known infectious agents, suchas viruses, bacteria, parasites, fungi, and the like. In variousembodiments, the peptide is selected from the list provided by Table 2.

For instance, non-limiting examples of a viruses from which antigensbearing epitopes that are incorporated in some embodiments into thedescribed peptides include, for instance, a vaccinia virus, a varicellazoster virus, a herpesvirus, e.g., herpes zoster virus orcytomegalovirus or Epstein-Barr virus, rubella, a hepatitis virus, e.g.,hepatitis A virus or hepatitis B virus or hepatitis C virus, influenza,e.g., type A or type B, a measles virus, a mumps virus, a polio virus, avariola (smallpox) virus, a rabies virus, a coronavirus, Dengue virus,an Ebola virus, a West Nile virus, a yellow fever virus, or a zikavirus.

For instance, non-limiting examples of a bacteria from which antigensbearing epitopes that are incorporated in some embodiments into thedescribed peptides include, for example, a Bordetella pertussis,chlamydia, trachomatis, Clostridium tetani, diphtheria, Hemophilusinfluenza, Meningococcus, pneumococcus, Vibrio cholera, Mycobacteriumtuberculosis, BCG, typhoid, E. coli, salmonella, Legionella pneumophila,rickettsia, Treponema pallidum pallidum, Streptococcus group A or groupB, Streptococcus pneumonia, Bacillus anthracis, Clostridium botulinum,or a Yersinia sp bacteria.

For instance, non-limiting examples of a parasite from which antigensbearing epitopes that are incorporated in some embodiments into thedescribed peptides include, Entamoeba histolytica, Toxoplasma gondii, aTrichinella sp., e.g., Trichinella spiralis, a Trichomonas sp., e.g.,Trichomonas vaginalis, a Trypanosoma sp., e.g., Trypanosoma bruceigambiense, Trypanosoma brucei rhodesiense, or a Trypanosoma cruzi, or aplasmodium, e.g., Plasmodium falciparum, Plasmodium vivax, or Plasmodiummalariae.

TABLE 2 Epitope Peptide Sequences SEQ ID Epitope Sequence NO Virus TypeMHC allele Viral Protein SLPRSRTPI 4 Chicken Pox A*02:01 IE62 (VZV)SAPLPSNRV 5 Chicken Pox A*02:01 IE62 (VZV) GSAPLPSNRV 6 Chicken PoxA*02:01 IE62 (VZV) ALWALPHAA 7 Chicken Pox A*02:01 IE62 (VZV) SLSGLYVFV8 Shingles A*02:01 Glycoprotein E vaccines YLGVYIWNM 9 Shingles A*02:01Glycoprotein E vaccines KIHEAPFDL 10 Shingles A*02:01 Glycoprotein Evaccines LLCLVIFLI 11 Shingles A*02:01 Glycoprotein E vaccines DLLLEWLYV12 Shingles A*02:01 Glycoprotein E vaccines SMYYAGLPV 13 ShinglesA*02:01 Glycoprotein E vaccines ILHDGGTTL 14 Shingles A*02:01Glycoprotein E vaccines WLYVPIDPT 15 Shingles A*02:01 Glycoprotein Evaccines VLMGFGIIT 16 Shingles A*02:01 Glycoprotein E vaccines CLVIFLICT17 Shingles A*02:01 Glycoprotein E vaccines KEADQPWIV 18 ShinglesA*02:01 Glycoprotein E vaccines VVSTVDHFV 19 Shingles A*02:01Glycoprotein E vaccines FLICTAKRM 20 Shingles A*02:01 Glycoprotein Evaccines VLRTEKQYL 21 Shingles A*02:01 Glycoprotein E vaccines HMWNYHSHV22 Shingles A*02:01 Glycoprotein E vaccines TVNKPVVGV 23 ShinglesA*02:01 Glycoprotein E vaccines FVVYFNGHV 24 Shingles A*02:01Glycoprotein E vaccines WIVVNTSTL 25 Shingles A*02:01 Glycoprotein Evaccines VAYTVVSTV 26 Shingles A*02:01 Glycoprotein E vaccines FMYMSLLGV27 measles A*02:01 m50 SLWGSLLML 28 measles A*02:01 C protein LLAVIFVMFL29 measles A*02:01 H38 SMYRVFEVGV 30 measles A*02:01 H250-259 ILPGQDLQYV31 measles A*02:01 H516-525 KLWCRHFCV 32 measles A*02:01 H576 KLWCRHFCVL33 measles A*02:01 H576 RLSDNGYYTV 34 measles A*02:01 M164 KLLRYYTEI 35measles A*02:01 F205 KLWESPQEI 36 measles A*02:01 C 84 RLLDRLVRL 37measles A*02:01 N50 KLMPNITLL 38 measles A*02:01 F57 TLLNNCTRV 39measles A*02:01 F64 EMLTLATWV 40 Hep B A*02:01 C64-72 FLPSDFFPSV 41Hep B A*02:01 Core 18 FLPADFFPSV 42 Hep B A*02:01 Core 19 FLPSDFFPSI 43Hep B A*02:01 Core 20 WLSLLVPF 44 Hep B A*02:01 ENV335FLLTRILTI or FLLTRILTL 45 Hep B A*02:01 ENV183 or 46 GLSPTVWLSV 47 Hep BA*02:01 ENV348 LLDYQGMLPV 48 Hep B A*02:01 ENV260 LLCLIFLLV 49 Hep BA*02:01 ENV251 SIVSPFIPLL 50 Hep B A*02:01 ENV370 FLLTKILTI 51 Hep BA*02:01 ENV183 ILSPFLPLL 52 Hep B A*02:01 ENV371 FLLSLGIHL 53 Hep BA*02:01 POL 575 GLSRYVARL 54 Hep B A*02:01 POL 455 SLYADSPSV 55 Hep BA*02:01 POL 816 YMDDVVLGA 56 Hep B A*02:01 POL 551 ALMPLYACI 57 Hep BA*02:01 POL 655 VLHKRTLGL 58 Hep B A*02:01 HBx 92 CLFKDWEEL 59 Hep BA*02:01 Hbx115 STLPETTVVRR 60 Hep B A*03, A*11 Core 141 EYLVSFGVW 61Hep B A*31, A*68 core 117 FFPSIRDLL 62 Hep B A*24 Core 23 SWLSLLVPF 63Hep B A*24 Env 334 KYTSFPWLL 64 Hep B A*24 Pol 756 HLSLRGLFV 65 Hep BA*02:01 HBx 52-60 CLFKDWEEL 66 Hep B A*02:01 HBx 115-123 LPSDFFPSV 67Hep B B*51 Core 19 GILGFVFTL 68 Influenza HLA-A2 M1ILGFVFTLTVPSERGLQRRRF 69 Influenza LIRHENRMVLASTTAKA 70 InfluenzaLQAYQKRMGVQMQR 71 Influenza YVYDHSGEAVK 72 Measles WLSLLVPFV 73 Hep B(K)GILGFVFTL(T)(V) 74 Influenza KLSTRGVQIASNEN 75 InfluenzaRGLQRRRFVQNALNGNG 76 Influenza FMYSDFHFI 77 Influenza NLVPMVATV 3Cytomegalovirus HLA-A2 VAIIEVDNEQPTTRAQKL 78 PoliovirusAny 9-mer sequence 79 Poliovirus of GACV AIIEVDNEQPTTRAQKLFAMWRITYKDTVQLRRKL SVRDRLARL 80 EBV LLDRVRFMGV 81 EBV CLGGLLTMV 82 EBVGLCTLVAML 83 EBV SVLGPISGHVLK 84 Cytomegalovirus HLA-A11 RPHERNGFTVL 85Cytomegalovirus HLA-B7 FTSQYRIQGKL 86 Cytomegalovirus HLA-A24YSEHPTFTSQY 87 Cytomegalovirus HLA-A1 EFFWDANDIY 88 CytomegalovirusHLA-B44 TTVYPPSSTAK 90 Cytomegalovirus HLA-A3 FVFPTKDVALR 91Cytomegalovirus HLA-A68 QTVTSTPVQGR 92 Cytomegalovirus HLA-A68PTFTSQYRIQGKL 93 Cytomegalovirus HLA-B38 FPTKDVAL 94 CytomegalovirusHLA-B35 SIINFEKL 95 RAHYNIVTF 96 SSPPMFRV 97 KLWAQCVQL 98 SARS-CoV-2A*02:01 KLPDDFTGCV 99 SARS-CoV-2 A*02:01 YLQPRTFLL 100 SARS-CoV-2A*02:01 LLYDANYFL 101 SARS-CoV-2 A*02:01 ALWEIQQVV 102 SARS-CoV-2A*02:01 LLLDRLNQL 103 SARS-CoV-2 A*02:01 YLFDESGEFKL 104 SARS-CoV-2A*02:01 FTSDYYQLY 105 SARS-CoV-2 A*01:01 PTDNYITTY 106 SARS-CoV-2A*01:01 ATSRTLSYY 107 SARS-CoV-2 A*01:01 CTDDNALAYY 108 SARS-CoV-2A*01:01 NTCDGTTFTY 109 SARS-CoV-2 A*01:01 DTDFVNEFY 110 SARS-CoV-2A*01:01 GTDLEGNFY 111 SARS-CoV-2 A*01:01 KTFPPTEPK 112 SARS-CoV-2A*03:01 KCYGVSPTK 113 SARS-CoV-2 A*03:01 VTNNTFTLK 114 SARS-CoV-2A*03:01 KTIQPRVEK 115 SARS-CoV-2 A*03:01 KTFPPTEPK 116 SARS-CoV-2A*11:01 VTDTPKGPK 117 SARS-CoV-2 A*11:01 ATEGALNTPK 118 SARS-CoV-2A*11:01 SARS-CoV-2 ASAFFGMSR 119 SARS-CoV-2 A*11:01 ATSRTLSYYK 120SARS-CoV-2 A*11:01 QYIKWPWYI 121 SARS-CoV-2 A*24:02 VYFLQSINF 122SARS-CoV-2 A*24:02 VYIGDPAQL 123 SARS-CoV-2 A*24:02 SPRWYFYYL 124SARS-CoV-2 B*07:02 RPDTRYVL 125 SARS-CoV-2 B*07:02 IPRRNVATL 126SARS-CoV-2 B*07:02

In various embodiments the epitope is found in one or more known humanvaccines, such as a childhood vaccine, early childhood, adolescent, orelderly (geriatric), vaccine. In various embodiments the vaccine is anearly childhood vaccine. Certain non-limiting examples of suitablevaccines from which such epitopes are found that are compatible with thedescribed peptides are listed in Table 3.

TABLE 3 Human Vaccines Containing Peptide-Compatible Epitopes CommercialResponsible National Type Name Form Source Regulatory AuthorityDiphtheria-Tetanus- Quinvaxem Liquid: Janssen Ministry of Food Pertussisready to use Vaccines and Drug Safety (whole cell)- Corp. Hepatitis B-Hemophilus influenzae type b Diphtheria-Tetanus Adsorbed Liquid: PT BioNational Agency of DT Vaccine ready to use Farma Drug and Food (Persero)Control Indonesia Diphtheria-Tetanus- DTP Liquid: PT Bio National Agencyof Pertussis Vaccine ready to use Farma Drug and Food (whole cell)(Persero) Control Indonesia Hepatitis B Hepatitis B Liquid: PT BioNational Agency of Vaccine ready to use Farma Drug and Food Recombinant(Persero) Control Indonesia Polio Vaccine - Oral Oral polio Liquid: PTBio National Agency of (OPV) Trivalent ready to use Farma Drug and Food(Persero) Control Indonesia Polio Vaccine - Oral Oral polio Liquid: PTBio National Agency of (OPV) Trivalent ready to use Farma Drug and Food(Persero) Control Indonesia Tetanus Toxoid TT vaccine Liquid: PT BioNational Agency of ready to use Farma Drug and Food (Persero) ControlIndonesia Tetanus Toxoid TT vaccine Liquid: PT Bio National Agency ofready to use Farma Drug and Food (Persero) Control Indonesia TetanusToxoid TT vaccine Liquid: PT Bio National Agency of ready to use FarmaDrug and Food (Persero) Control Indonesia Measles Measles Lyophilized PTBio National Agency of vaccine Lyophilized Farma Drug and Food active(Persero) Control Indonesia component to be reconstituted with excipientdiluent before use Yellow Fever Yellow Fever Lyophilized Bio- AgenciaNacional Lyophilized Manguinhos/ da Vigilancia active Fiocruz Sanitariacomponent to be reconstituted with excipient diluent before use YellowFever Yellow Fever Lyophilized Bio- Agencia Nacional LyophilizedManguinhos/ da Vigilancia active Fiocruz Sanitaria component to bereconstituted with excipient diluent before use Yellow Fever YellowFever Lyophilized Bio- Agencia Nacional Lyophilized Manguinhos/ daVigilancia active Fiocruz Sanitaria component to be reconstituted withexcipient diluent before use Hepatitis B Heberbiovac Liquid: Centro deCentro para el HB ready to use Ingenieria Control Estatal de Genetica yla Calidad de los Biotecnologia Medicamentos Hepatitis B HeberbiovacLiquid: Centro de Centro para el HB ready to use Ingenieria ControlEstatal de Genetica y la Calidad de los Biotecnologia MedicamentosRabies Rabipur Lyophilized Chiron Central Drugs active Behring StandardControl component Vaccines Organization to be Private Ltd. reconstitutedwith excipient diluent before use Rabies Rabipur LyophilizedGlaxoSmithKline Paul-Ehrlich- active Vaccines Institut component GmbH tobe reconstituted with excipient diluent before use Haemophilus Vaxem HIBLiquid: Novartis Agenzia Italiana influenzae type b ready to useVaccines del Farmaco and Diagnostics S.r.l Hepatitis B Engerix Liquid:GlaxoSmithKline Federal Agency for ready to use Biologicals Medicinesand SA Health Products Hepatitis B Engerix Liquid: GlaxoSmithKlineFederal Agency for ready to use Biologicals Medicines and SA HealthProducts Hepatitis B Engerix Liquid: GlaxoSmithKline Federal Agency forready to use Biologicals Medicines and SA Health Products PolioVaccine - Oral Polio sabin Liquid: GlaxoSmithKline Federal Agency for(OPV) Trivalent ready to use Biologicals Medicines and SA HealthProducts Polio Vaccine - Oral Polio sabin Liquid: GlaxoSmithKlineFederal Agency for (OPV) Trivalent ready to use Biologicals Medicinesand SA Health Products Measles, Priorix Lyophilized GlaxoSmithKlineFederal Agency for Mumps and active Biologicals Medicines and Rubellacomponent SA Health Products to be reconstituted with excipient diluentbefore use Rotavirus Rotarix Liquid: GlaxoSmithKline Federal Agency forready to use Biologicals Medicines and SA Health Products PolioVaccine - Oral Polioviral Liquid: Haffkine Central Drugs (OPV) Trivalentvaccine ready to use Bio Standard Control Pharmaceutical OrganizationCorporation Ltd Yellow Fever Stabilized Lyophilized Institut Ministèrede la Yellow Fever active Pasteur de Santé publique Vaccine componentDakar to be reconstituted with excipient diluent before use Yellow FeverStabilized Lyophilized Institut Ministère de la Yellow Fever activePasteur de Santé publique Vaccine component Dakar to be reconstitutedwith excipient diluent before use Yellow Fever Stabilized LyophilizedInstitut Ministère de la Yellow Fever active Pasteur de Santé publiqueVaccine component Dakar to be reconstituted with excipient diluentbefore use BCG BCG Freeze Lyophilized Japan BCG Chiba Local Dried activeLaboratory Government Glutamate component vaccine to be reconstitutedwith excipient diluent before use Hepatitis B Euvax B Liquid: LG ChemMinistry of Food ready to use Ltd and Drug Safety Hepatitis B Euvax BLiquid: LG Chem Ministry of Food ready to use Ltd and Drug Safety BCGBCG Lyophilized Bul Bio - Bulgarian Drug Vaccine active National Agencycomponent Center of to be Infectious reconstituted and with Parasiticexcipient Diseases diluent Ltd. before use BCG BCG Lyophilized Bul Bio -Bulgarian Drug Vaccine active National Agency component Center of to beInfectious reconstituted and with Parasitic excipient Diseases diluentLtd. before use Tetanus Toxoid Tetatox Liquid: Bul Bio - Bulgarian Drugready to use National Agency Center of Infectious and Parasitic DiseasesLtd. Tetanus Toxoid Tetatox Liquid: Bul Bio - Bulgarian Drug ready touse National Agency Center of Infectious and Parasitic Diseases Ltd.Diphtheria-Tetanus Diftet Liquid: Bul Bio - Bulgarian Drug ready to useNational Agency Center of Infectious and Parasitic Diseases Ltd.Diphtheria-Tetanus Diftet Liquid: Bul Bio - Bulgarian Drug ready to useNational Agency Center of Infectious and Parasitic Diseases Ltd.Diphtheria-Tetanus Tetadif Liquid: Bul Bio - Bulgarian Drug (reducedantigen ready to use National Agency content) Center of Infectious andParasitic Diseases Ltd. Diphtheria-Tetanus Tetadif Liquid: Bul Bio -Bulgarian Drug (reduced antigen ready to use National Agency content)Center of Infectious and Parasitic Diseases Ltd. Diphtheria-Tetanus-Easyfive-TT Liquid: Panacea Central Drugs Pertussis ready to use BiotecLtd. Standard Control (whole cell)- Organization Hepatitis B-Haemophilus influenzae type b Diphtheria-Tetanus IMOVAX Liquid: SanofiAgence nationale (reduced antigen dT adult ready to use Pasteur SA desécurite du content) médicament et des produits de santé Polio Vaccine -IMOVAX Liquid: Sanofi Agence nationale Inactivated (IPV) POLIO ready touse Pasteur SA de sécurité du médicament et des produits de santé PolioVaccine - Oral OPVERO Liquid: Sanofi Agence nationale (OPV) Trivalentready to use Pasteur SA de sécurité du médicament et des produits desanté Polio Vaccine - Oral OPVERO Liquid: Sanofi Agence nationale (OPV)Trivalent ready to use Pasteur SA de sécurité du médicament et desproduits de santé Polio Vaccine - Oral OPVERO Liquid: Sanofi Agencenationale (OPV) Trivalent ready to use Pasteur SA de sécurité dumédicament et des produits de santé Tetanus Toxoid TETAVAX Liquid:Sanofi Agence nationale ready to use Pasteur SA de sécurité dumédicament et des produits de santé Tetanus Toxoid TETAVAX Liquid:Sanofi Agence nationale ready to use Pasteur SA de sécurité dumédicament et des produits de santé Haemophilus Act-HIB LyophilizedSanofi Agence nationale influenzae type b active Pasteur SA de sécuritédu component médicament et des to be produits de santé reconstitutedwith excipient diluent before use Rabies VERORAB Lyophilized SanofiAgence nationale active Pasteur SA de sécurité du component médicamentet des to be produits de santé reconstituted with excipient diluentbefore use Yellow Fever STAMARIL Lyophilized Sanofi Agence nationaleactive Pasteur SA de sécurité du component médicament et des to beproduits de santé reconstituted with excipient diluent before useMeningococcal POLYSACCHARIDE Lyophilized Sanofi Agence nationale A + CMENINGOCOCCAL active Pasteur SA de sécurité du A + C VACCINE componentmédicament et des to be produits de santé reconstituted with excipientdiluent before use Polio Vaccine - Oral ORAL Liquid: Sanofi Agencenationale (OPV) Monovalent MONOVALENT ready to use Pasteur SA desécurité du Type 1 TYPE 1 médicament et des POLIOMYELITIS produits desanté VACCINE cholera: inactivated Dukoral Liquid: Valneva MedicalProducts oral ready to use Sweden Agency AB BCG BCG Lyophilized SerumCentral Drugs Vaccine active Institute of Standard Control componentIndia Pvt. Organization to be Ltd. reconstituted with excipient diluentbefore use Diphtheria-Tetanus Diphtheria Liquid: Serum Central Drugs andTetanus ready to use Institute of Standard Control Vaccine India Pvt.Organization Adsorbed Ltd. (Paediatric) Diphtheria-Tetanus DiphtheriaLiquid: Serum Central Drugs and Tetanus ready to use Institute ofStandard Control Vaccine India Pvt. Organization Adsorbed Ltd.(Pediatric) Diphtheria-Tetanus Diphtheria Liquid: Serum Central Drugsand Tetanus ready to use Institute of Standard Control Vaccine IndiaPvt. Organization Adsorbed Ltd. (Pediatric) Diphtheria-TetanusDiphtheria Liquid: Serum Central Drugs (reduced antigen and Tetanusready to use Institute of Standard Control content) Vaccine India Pvt.Organization Adsorbed for Ltd. Adults and Adolescents Diphtheria-TetanusDiphtheria Liquid: Serum Central Drugs (reduced antigen and Tetanusready to use Institute of Standard Control content) Vaccine India Pvt.Organization Adsorbed for Ltd. 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Vaccine Adsorbed Diphtheria-Tetanus- Diphtheria,Liquid: Serum Central Drugs Pertussis Tetanus, ready to use Institute ofStandard Control (whole cell)- Pertussis and India Pvt. OrganizationHepatitis B Hepatitis B Ltd. Vaccine Adsorbed Diphtheria-Tetanus-Diphtheria, Liquid: Serum Central Drugs Pertussis Tetanus, ready to useInstitute of Standard Control (whole cell)- Pertussis and India Pvt.Organization Hepatitis B Hepatitis B Ltd. Vaccine Adsorbed Hepatitis BHepatitis B Liquid: Serum Central Drugs Vaccine ready to use Instituteof Standard Control (rDNA) India Pvt. Organization (Adult) Ltd.Hepatitis B Hepatitis B Liquid: Serum Central Drugs Vaccine ready to useInstitute of Standard Control (rDNA) India Pvt. Organization (Adult)Ltd. Hepatitis B Hepatitis B Liquid: Serum Central Drugs Vaccine readyto use Institute of Standard Control (rDNA) India Pvt. Organization(Paediatric) Ltd. 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Attenuated reconstituted withexcipient diluent before use Measles and Measles and Lyophilized SerumCentral Drugs Rubella Rubella active Institute of Standard ControlVaccine, component India Pvt. Organization Live, to be Ltd. Attenuatedreconstituted with excipient diluent before use Measles and Measles andLyophilized Serum Central Drugs Rubella Rubella active Institute ofStandard Control Vaccine, component India Pvt. Organization Live, to beLtd. Attenuated reconstituted with excipient diluent before use Measles,Measles, Lyophilized Serum Central Drugs Mumps and Mumps and activeInstitute of Standard Control Rubella Rubella component India Pvt.Organization Vaccine, to be Ltd. Live, reconstituted Attenuated withexcipient diluent before use Measles, Measles, Lyophilized Serum CentralDrugs Mumps and Mumps and active Institute of Standard Control RubellaRubella component India Pvt. Organization Vaccine, to be Ltd. Live,reconstituted Attenuated with excipient diluent before use Measles,Measles, Lyophilized Serum Central Drugs Mumps and Mumps and activeInstitute of Standard Control Rubella Rubella component India Pvt.Organization Vaccine, to be Ltd. Live, reconstituted Attenuated withexcipient diluent before use Measles, Measles, Lyophilized Serum CentralDrugs Mumps and Mumps and active Institute of Standard Control RubellaRubella component India Pvt. Organization Vaccine, to be Ltd. Live,reconstituted Attenuated with excipient diluent before use MeaslesMeasles Lyophilized Serum Central Drugs Vaccine, active Institute ofStandard Control Live, component India Pvt. Organization Attenuated tobe Ltd. reconstituted with excipient diluent before use Measles MeaslesLyophilized Serum Central Drugs Vaccine, active Institute of StandardControl Live, component India Pvt. Organization Attenuated to be Ltd.reconstituted with excipient diluent before use Measles MeaslesLyophilized Serum Central Drugs Vaccine, active Institute of StandardControl Live, component India Pvt. Organization Attenuated to be Ltd.reconstituted with excipient diluent before use Measles MeaslesLyophilized Serum Central Drugs Vaccine, active Institute of StandardControl Live, component India Pvt. Organization Attenuated to be Ltd.reconstituted with excipient diluent before use Rubella RubellaLyophilized Serum Central Drugs Vaccine, active Institute of StandardControl Live, component India Pvt. Organization Attenuated to be Ltd.reconstituted with excipient diluent before use Rubella RubellaLyophilized Serum Central Drugs Vaccine, active Institute of StandardControl Live, component India Pvt. Organization Attenuated to be Ltd.reconstituted with excipient diluent before use Rubella RubellaLyophilized Serum Central Drugs Vaccine, active Institute of StandardControl Live, component India Pvt. Organization Attenuated to be Ltd.reconstituted with excipient diluent before use Rubella RubellaLyophilized Serum Central Drugs Vaccine, active Institute of StandardControl Live, component India Pvt. Organization Attenuated to be Ltd.reconstituted with excipient diluent before use Tetanus Toxoid ShanTTLiquid: Shantha Central Drugs ready to use Biotechnics Standard ControlPrivate Organization Limited (A Sanofi Company) Tetanus Toxoid ShanTTLiquid: Shantha Central Drugs ready to use Biotechnics Standard ControlPrivate Organization Limited (A Sanofi Company) Diphtheria-Tetanus-Shan-5 Liquid: Shantha Central Drugs Pertussis ready to use BiotechnicsStandard Control (whole cell)- Private Organization Hepatitis B- Limited(A Haemophilus Sanofi influenzae type b Company) Diphtheria-Tetanus-Shan-5 Liquid: Shantha Central Drugs Pertussis ready to use BiotechnicsStandard Control (whole cell)- Private Organization Hepatitis B- Limited(A Haemophilus Sanofi influenzae type b Company) BCG BCG Lyophilized AJDanish Medicines Vaccine SSI active Vaccines Agency component A/S to bereconstituted with excipient diluent before use Rotavirus RotateqLiquid: Merck CBER/FDA ready to use Vaccines Measles, rHA M-M-RLyophilized Merck European Mumps and II active Vaccines Medicines AgencyRubella component to be reconstituted with excipient diluent before useRotavirus Rotarix Liquid: GlaxoSmithKline Federal Agency for ready touse Biologicals Medicines and SA Health Products Yellow Fever —Lyophilized Federal Federal Service on active State Surveillance incomponent Budgetary Healthcare to be Scientific (ROSZDRAVNADZOR)reconstituted Institution of the Russian Federation with «Chumakovexcipient Federal diluent Scientific before use Center for Reserch &Development of Immune- And Biological Products», Russian Academy ofSciences Yellow Fever — Lyophilized Federal Federal Service on activeState Surveillance in component Budgetary Healthcare to be Scientific(ROSZDRAVNADZOR) reconstituted Institution of the Russian Federationwith «Chumakov excipient Federal diluent Scientific before use Centerfor Reserch & Development of Immune- And Biological Products», RussianAcademy of Sciences Yellow Fever — Lyophilized Federal Federal Serviceon active State Surveillance in component Budgetary Healthcare to beScientific (ROSZDRAVNADZOR) reconstituted Institution of the RussianFederation with «Chumakov excipient Federal diluent Scientific beforeuse Center for Reserch & Development of Immune- And BiologicalProducts», Russian Academy of Sciences Human Gardasil Liquid: MerckEuropean Papillomavirus ready to use Vaccines Medicines Agency(Quadrivalent) Human Cervarix Liquid: GlaxoSmithKline Federal Agency forPapillomavirus ready to use Biologicals Medicines and (Bivalent) SAHealth Products Human Cervarix Liquid: GlaxoSmithKline Federal Agencyfor Papillomavirus ready to use Biologicals Medicines and (Bivalent) SAHealth Products Polio Vaccine - Oral Polio Sabin Liquid: GlaxoSmithKlineFederal Agency for (OPV) Monovalent Mono T1 ready to use BiologicalsMedicines and Type 1 SA Health Products Polio Vaccine - Oral Polio SabinLiquid: GlaxoSmithKline Federal Agency for (OPV) Monovalent Mono T1ready to use Biologicals Medicines and Type 1 SA Health Products PolioVaccine - Oral Polio Sabin Liquid: GlaxoSmithKline Federal Agency for(OPV) Bivalent Types One and ready to use Biologicals Medicines and 1and 3 Three SA Health Products Polio Vaccine - Oral Polio Sabin Liquid:GlaxoSmithKline Federal Agency for (OPV) Bivalent Types One and ready touse Biologicals Medicines and 1 and 3 Three SA Health ProductsHaemophilus Haemophilus Lyophilized Serum Central Drugs influenzae typeb influenzae type b active Institute of Standard Control Conjugatecomponent India Pvt. Organization Vaccine to be Ltd. reconstituted withexcipient diluent before use Polio Vaccine - Oral Monovalent Liquid:Haffkine Central Drugs (OPV) Monovalent type 1 Oral ready to use BioStandard Control Type 1 Poliomyelitis Pharmaceutical Organizationvaccine, IP Corporation (mOPV1) Ltd Polio Vaccine - Oral MonovalentLiquid: PT Bio National Agency of (OPV) Monovalent Oral ready to useFarma Drug and Food Type 1 Poliomyelitis (Persero) Control IndonesiaVaccine Type 1 (mOPV1) Tetanus Toxoid None used Liquid: BiologicalCentral Drugs on labelling ready to use E. Limited Standard Control forsupply Organization through UN agencies. Also marketed with labelledcommercial name BEtt. Pneumococcal Synflorix Liquid: GlaxoSmithKlineEuropean (conjugate) ready to use Biologicals Medicines Agency SADiphtheria-Tetanus- Diphtheria, Lyophilized Serum Central DrugsPertussis Tetanus, active Institute of Standard Control (whole cell)-Pertussis, component India Pvt. Organization Hepatitis B- Hepatitis Band to be Ltd. Haemophilus Haemophilus reconstituted influenzae type binfluenzae type b with liquid Conjugate active Vaccine component beforeuse Polio Vaccine - Oral Bivalent Liquid: PT Bio National Agency of(OPV) Bivalent Types Oral ready to use Farma Drug and Food 1 and 3Poliomyelitis (Persero) Control Indonesia Vaccine Type 1&3 (bOPV 1&3)Meningococcal A Meningococcal A Lyophilized Serum Central DrugsConjugate 10 μg Conjugate active Institute of Standard ControlMenAfriVac component India Pvt. Organization to be Ltd. reconstitutedwith excipient diluent before use Haemophilus Quimi-Hib Liquid: Centrode Centro para el influenzae type b ready to use Ingenieria ControlEstatal de Genetica y la Calidad de los Biotecnologia MedicamentosPneumococcal Synflorix Liquid: GlaxoSmithKline European (conjugate)ready to use Biologicals Medicines Agency SA Influenza, seasonalFluvirin Liquid: Seqirus CBER/FDA ready to use Vaccines Limited PolioVaccine - Oral Bivalent type Liquid: Haffkine Central Drugs (OPV)Bivalent Types 1&3 Oral ready to use Bio Standard Control 1 and 3Poliomyelitis Pharmaceutical Organization vaccine, IP Corporation(bOPV1&3) Ltd Influenza, seasonal Fluzone Liquid: Sanofi CBER/FDA readyto use Pasteur- USA Influenza, seasonal Fluzone Liquid: Sanofi CBER/FDAready to use Pasteur- USA Diphtheria-Tetanus- Diphtheria, LyophilizedSerum Central Drugs Pertussis Tetanus, active Institute of StandardControl (whole cell)- Pertussis, component India Pvt. OrganizationHepatitis B- Hepatitis B and to be Ltd. Haemophilus Haemophilusreconstituted influenzae type b influenzae type b with liquid Conjugateactive Vaccine component before use Influenza, seasonal GC FLU Liquid:Green Ministry of Food Multi inj. ready to use Cross and Drug SafetyCorporation Diphtheria-Tetanus- Diphtheria, Lyophilized Serum CentralDrugs Pertussis Tetanus, active Institute of Standard Control (wholecell)- Pertussis, component India Pvt. Organization Hepatitis B-Hepatitis B and to be Ltd. Haemophilus Haemophilus reconstitutedinfluenzae type b influenzae type b with liquid Conjugate active Vaccinecomponent before use Influenza, pandemic Panvax Liquid: SeqirusTherapeutic Goods H1N1 ready to use Limited Administration Influenza,pandemic Green Flu-S Liquid: Green Ministry of Food H1N1 ready to useCross and Drug Safety Corporation Influenza, pandemic Influenza ALiquid: MedImmune CBER/FDA H1N1 (H1N1) 2009 ready to use monovalentvaccine Influenza, pandemic Celtura Liquid: Seqirus Paul-Ehrlich- H1N1ready to use GmbH Institut Influenza, pandemic Focetria Liquid: SeqirusH1N1 ready to use Vaccines Limited Influenza, pandemic Fluvirin- Liquid:Seqirus CBER/FDA H1N1 H1N1 ready to use Vaccines Limited Influenza,pandemic Panenza Liquid: Sanofi Agence nationale H1N1 ready to usePasteur SA de sécurité du médicament et des produits de santé Influenza,pandemic Influenza A Liquid: Sanofi CBER/FDA H1N1 (H1N1) 2009 ready touse Pasteur- monovalent USA vaccine Influenza, pandemic Influenza ALiquid: Sanofi CBER/FDA H1N1 (H1N1) 2009 ready to use Pasteur-monovalent USA vaccine Diphtheria-Tetanus- Diphtheria, Lyophilized SerumCentral Drugs Pertussis Tetanus, active Institute of Standard Control(whole cell)- Pertussis and component India Pvt. OrganizationHaemophilus Haemophilus to be Ltd. influenzae type b influenzae type breconstituted Conjugate with liquid Vaccine active component before usePolio Vaccine - Poliorix Liquid: GlaxoSmithKline Federal Agency forInactivated (IPV) ready to use Biologicals Medicines and SA HealthProducts Polio Vaccine - Poliorix Liquid: GlaxoSmithKline Federal Agencyfor Inactivated (IPV) ready to use Biologicals Medicines and SA HealthProducts Pneumococcal Prevenar 13 Liquid: Pfizer European (conjugate)ready to use Medicines Agency Diphtheria-Tetanus- Diphtheria, Liquid:Serum Central Drugs Pertussis Tetanus, ready to use Institute ofStandard Control (whole cell)- Pertussis, India Pvt. OrganizationHepatitis B- Hepatitis B and Ltd. Haemophilus Haemophilus influenzaetype b influenzae type b Conjugate Vaccine Adsorbed Diphtheria-Tetanus-Diphtheria, Liquid: Serum Central Drugs Pertussis Tetanus, ready to useInstitute of Standard Control (whole cell)- Pertussis, India Pvt.Organization Hepatitis B- Hepatitis B and Ltd. Haemophilus Haemophilusinfluenzae type b influenzae type b Conjugate Vaccine AdsorbedDiphtheria-Tetanus- Diphtheria, Liquid: Serum Central Drugs PertussisTetanus, ready to use Institute of Standard Control (whole cell)-Pertussis, India Pvt. Organization Hepatitis B- Hepatitis B and Ltd.Haemophilus Haemophilus influenzae type b influenzae type b ConjugateVaccine Adsorbed Polio Vaccine - Oral Polio Sabin Liquid:GlaxoSmithKline Federal Agency for (OPV) Monovalent Mono Three ready touse Biologicals Medicines and Type 3 (oral) SA Health Products PolioVaccine - Oral Polio Sabin Liquid: GlaxoSmithKline Federal Agency for(OPV) Monovalent Mono Three ready to use Biologicals Medicines and Type3 (oral) SA Health Products Polio Vaccine - Poliomyelitis Liquid:Bilthoven Medicines Inactivated (IPV) vaccine ready to use BiologicalsEvaluation Board (MEB) Polio Vaccine - IPV Vaccine Liquid: AJ DanishMedicines Inactivated (IPV) SSI ready to use Vaccines Agency A/SInfluenza, seasonal GC FLU inj Liquid: Green Ministry of Food ready touse Cross and Drug Safety Corporation Polio Vaccine - Oral Polio SabinLiquid: GlaxoSmithKline Federal Agency for (OPV) Monovalent Mono Twoready to use Biologicals Medicines and Type 2 (oral) SA Health ProductsPolio Vaccine - Oral Polio Sabin Liquid: GlaxoSmithKline Federal Agencyfor (OPV) Monovalent Mono Two ready to use Biologicals Medicines andType 2 (oral) SA Health Products Typhoid Typhim-Vi Liquid: Sanofi Agencenationale (Polysaccharide) ready to use Pasteur SA de sécurité dumédicament et des produits de santé Influenza, seasonal Vaxigrip Liquid:Sanofi Agence nationale ready to use Pasteur SA de sécurité dumédicament et des produits de santé Polio Vaccine - Oral BIOPOLIOLiquid: Bharat Central Drugs (OPV) Bivalent Types B1/3 ready to useBiotech Standard Control 1 and 3 International Organization LimitedDiphtheria-Tetanus none Liquid: PT Bio National Agency of (reducedantigen ready to use Farma Drug and Food content) (Persero) ControlIndonesia Polio Vaccine - Oral none Liquid: Sanofi Agence nationale(OPV) Bivalent Types ready to use Pasteur SA de sécurité du 1 and 3médicament et des produits de santé Diphtheria-Tetanus- None usedLyophilized Biological Central Drugs Pertussis on labelling active E.Limited Standard Control (whole cell)- for supply component OrganizationHepatitis B- through UN to be Haemophilus agencies. reconstitutedinfluenzae type b Also with liquid marketed active with labelledcomponent commercial before use name ComBE Five (Reconstituted).Diphtheria-Tetanus- None used Lyophilized Biological Central DrugsPertussis on labelling active E. Limited Standard Control (whole cell)-for supply component Organization Hepatitis B- through UN to beHaemophilus agencies. reconstituted influenzae type b Also with liquidmarketed active with labelled component commercial before use name ComBEFive (Reconstituted). cholera: inactivated Shanchol Liquid: ShanthaCentral Drugs oral ready to use Biotechnics Standard Control PrivateOrganization Limited (A Sanofi Company) Measles, Priorix LyophilizedGlaxoSmithKline Federal Agency for Mumps and active BiologicalsMedicines and Rubella component SA Health Products to be reconstitutedwith excipient diluent before use Measles Measles Lyophilized PT BioNational Agency of vaccine active Farma Drug and Food component(Persero) Control Indonesia to be reconstituted with excipient diluentbefore use Polio Vaccine - Oral Poliomyelitis Liquid: Serum CentralDrugs (OPV) Bivalent Types Vaccine ready to use Institute of StandardControl 1 and 3 (Oral), India Pvt. Organization Bivalent Ltd. types 1and 3 Influenza, pandemic NASOVAC Lyophilized Serum Central Drugs H1N1Influenza active Institute of Standard Control Vaccine, component IndiaPvt. Organization Live to be Ltd. Attenuated reconstituted (Human) withFreeze-Dried excipient diluent before use Influenza, pandemic NASOVACLyophilized Serum Central Drugs H1N1 Influenza active Institute ofStandard Control Vaccine, component India Pvt. Organization Live to beLtd. Attenuated reconstituted (Human) with Freeze-Dried excipientdiluent before use Tetanus Toxoid None used Liquid: Biological CentralDrugs on labelling ready to use E. Limited Standard Control for supplyOrganization through UN agencies. Also marketed with labelled commercialname BEtt. Tetanus Toxoid None used Liquid: Biological Central Drugs onlabelling ready to use E. Limited Standard Control for supplyOrganization through UN agencies. Also marketed with labelled commercialname BEtt. Japanese JEEV ® Liquid: Biological Central Drugs Encephalitisready to use E. Limited Standard Control Vaccine Organization(Inactivated) 6 μg Hepatitis A (Human Havrix 1440 Liquid:GlaxoSmithKline Federal Agency for Diploid Cell), Adult ready to useBiologicals Medicines and Inactivated (Adult) SA Health ProductsHepatitis A (Human Havrix 720 Liquid: GlaxoSmithKline Federal Agency forDiploid Cell), Junior ready to use Biologicals Medicines and InactivatedSA Health Products (Paediatric) Diphtheria-Tetanus- Boostrix Liquid:GlaxoSmithKline Federal Agency for Pertussis ready to use BiologicalsMedicines and (acellular) SA Health Products Meningococcal MenveoLyophilized GlaxoSmithKline European ACYW-135 active Vaccines MedicinesAgency (conjugate vaccine) component S.r.l. to be reconstituted withliquid active component before use Meningococcal Menactra Liquid: SanofiCBER/FDA ACYW-135 ready to use Pasteur- (conjugate vaccine) USADiphtheria-Tetanus- Easyfive-TT Liquid: Panacea Central Drugs Pertussisready to use Biotec Ltd. Standard Control (whole cell)- OrganizationHepatitis B- Haemophilus influenzae type b Japanese Japanese LyophilizedChengdu National Medical Encephalitis Encephalitis active Institute ofProducts Vaccine (live, Vaccine Live component Biological Administrationattenuated) (SA14-14-2) to be Products reconstituted Co., Ltd withexcipient diluent before use Japanese Japanese Lyophilized ChengduNational Medical Encephalitis Encephalitis active Institute of ProductsVaccine (live, Vaccine Live component Biological Administrationattenuated) (SA14-14-2) to be Products reconstituted Co., Ltd withexcipient diluent before use Diphtheria-Tetanus- None used Liquid:Biological Central Drugs Pertussis on labelling ready to use E. LimitedStandard Control (whole cell)- for supply Organization Hepatitis B-through UN Haemophilus agencies. influenzae type b Also marketed withlabelled commercial name ComBE Five (Liquid). Diphtheria-Tetanus- Noneused Liquid: Biological Central Drugs Pertussis on labelling ready touse E. Limited Standard Control (whole cell)- for supply OrganizationHepatitis B- through UN Haemophilus agencies. influenzae type b Alsomarketed with labelled commercial name ComBE Five (Liquid). JapaneseIMOJEV Lyophilized GPO-MBP Thai Food and Encephalitis MD active Co.,Ltd. Drug Vaccine (live, component Administration attenuated) to bereconstituted with excipient diluent before use Diphtheria-Tetanus- Noneused Liquid: Biological Central Drugs Pertussis on labelling ready touse E. Limited Standard Control (whole cell) for supply Organizationthrough UN agencies. Also marketed with labelled commercial name TRIPVACDiphtheria-Tetanus- None used Liquid: Biological Central Drugs Pertussison labelling ready to use E. Limited Standard Control (whole cell) forsupply Organization through UN agencies. Also marketed with labelledcommercial name TRIPVAC Diphtheria-Tetanus None used Liquid: BiologicalCentral Drugs (reduced antigen on labelling ready to use E. LimitedStandard Control content) for supply Organization through UN agencies.Also marketed with labelled commercial name BE Td Diphtheria-TetanusNone used Liquid: Biological Central Drugs (reduced antigen on labellingready to use E. Limited Standard Control content) for supplyOrganization through UN agencies. Also marketed with labelled commercialname BE Td Polio Vaccine - Oral Poliomyelitis Liquid: Serum CentralDrugs (OPV) Bivalent Types Vaccine ready to use Institute of StandardControl 1 and 3 (Oral), India Pvt. Organization Bivalent Ltd. types 1and 3 Polio Vaccine - Poliomyelitis Liquid: Bilthoven MedicinesInactivated (IPV) vaccine ready to use Biologicals Evaluation Boardmultidose, (MEB) suspension for injection 2.5 mL Influenza, seasonalNasovac-S Lyophilized Serum Central Drugs Influenza active Institute ofStandard Control Vaccine, component India Pvt. Organization Live, to beLtd. Attenuated reconstituted (Human) with excipient diluent before useDiphtheria-Tetanus- Hexaxim Liquid: Sanofi European Pertussis ready touse Pasteur SA Medicines Agency (acellular)- Hepatitis B- Haemophilusinfluenzae type b- Polio (Inactivated) Meningococcal A Meningococcal ALyophilized Serum Central Drugs Conjugate 5 μg Conjugate 5 activeInstitute of Standard Control micrograms component India Pvt.Organization MenAfriVac to be Ltd. 5 μg reconstituted with excipientdiluent before use Diphtheria-Tetanus- None used Liquid: BiologicalCentral Drugs Pertussis on labelling ready to use E. Limited StandardControl (whole cell)- for supply Organization Hepatitis B- through UNHaemophilus agencies. influenzae type b Also marketed with labelledcommercial name ComBE Five (Liquid). Diphtheria-Tetanus- None usedLiquid: Biological Central Drugs Pertussis on labelling ready to use E.Limited Standard Control (whole cell)- for supply Organization HepatitisB- through UN Haemophilus agencies. influenzae type b Also marketed withlabelled commercial name ComBE Five (Liquid). Polio Vaccine -Poliomyelitis Liquid: Serum Central Drugs Inactivated (IPV) Vaccineready to use Institute of Standard Control (Inactivated) India Pvt.Organization Ltd. Polio Vaccine - Oral BIOPOLIO Liquid: Bharat CentralDrugs (OPV) Trivalent ready to use Biotech Standard ControlInternational Organization Limited Polio Vaccine - Oral BIOPOLIO Liquid:Bharat Central Drugs (OPV) Trivalent ready to use Biotech StandardControl International Organization Limited Influenza, seasonal InfluenzaLiquid: Hualan National Medical Vaccine ready to use Biological Products(Split virion, Bacterin Administration inactivated) Co., Ltd Influenza,seasonal IL-YANG Liquid: IL-YANG Ministry of Food FLU ready to usePHARMACEUTICAL and Drug Safety Vaccine INJ. CO., LTD. BCG BCG vaccineLyophilized GreenSignal Central Drugs (Freeze active Bio StandardControl Dried) - component Pharma Organization Intradermal to be Limitedreconstituted with excipient diluent before use Influenza, seasonalFluzone Liquid: Sanofi CBER/FDA Quadrivalent Quadrivalent ready to usePasteur- USA Influenza, seasonal Fluzone Liquid: Sanofi CBER/FDAQuadrivalent Quadrivalent ready to use Pasteur- USA Polio Vaccine - OralBivalent Liquid: PT Bio National Agency of (OPV) Bivalent Types Oralready to use Farma Drug and Food 1 and 3 Poliomyelitis (Persero) ControlIndonesia Vaccine Type 1&3 (bOPV 1&3) cholera: inactivated EuvicholLiquid: EuBiologics Ministry of Food oral ready to use Co., Ltd. andDrug Safety Polio Vaccine - Oral ORAL Liquid: Sanofi Agence nationale(OPV) Monovalent MONOVALENT ready to use Pasteur SA de sécurité du Type2 TYPE 2 médicament et des POLIOMYELITIS produits de santé VACCINE(mOPV2) Polio Vaccine - Oral ORAL Liquid: Sanofi Agence nationale (OPV)Monovalent MONOVALENT ready to use Pasteur SA de sécurité du Type 3 TYPE3 médicament et des POLIOMYELITIS produits de santé VACCINEMeningococcal Nimenrix Lyophilized Pfizer European ACYW-135 activeMedicines Agency (conjugate vaccine) component to be reconstituted withexcipient diluent before use Diphtheria-Tetanus- Eupenta Liquid: LG ChemMinistry of Food Pertussis ready to use Ltd and Drug Safety (wholecell)- Hepatitis B- Haemophilus influenzae type b Diphtheria-Tetanus-Eupenta Liquid: LG Chem Ministry of Food Pertussis ready to use Ltd andDrug Safety (whole cell)- Hepatitis B- Haemophilus influenzae type bHuman Gardasil 9 Liquid: Merck European Papillomavirus ready to useVaccines Medicines Agency (Ninevalent) Influenza, seasonal GCFLU Liquid:Green Ministry of Food Quadrivalent Quadrivalent ready to use Cross andDrug Safety inj. Corporation Diphtheria-Tetanus- Pentabio Liquid: PT BioNational Agency of Pertussis ready to use Farma Drug and Food (wholecell)- (Persero) Control Indonesia Hepatitis B- Haemophilus influenzaetype b Diphtheria-Tetanus- Pentabio Liquid: PT Bio National Agency ofPertussis ready to use Farma Drug and Food (whole cell)- (Persero)Control Indonesia Hepatitis B- Haemophilus influenzae type b Hepatitis A(Human HEALIVE Liquid: Sinovac National Medical Diploid Cell), ready touse Biotech Products Inactivated (Adult) Co. Ltd AdministrationVaricella Varivax Lyophilized Merck CBER/FDA active Vaccines componentto be reconstituted with excipient diluent before use Rotavirus (live,Rotavac Liquid: Bharat Central Drugs attenuated) ready to use BiotechStandard Control International Organization Limited Diphtheria-Tetanus-Adacel Liquid: Sanofi Health Canada - Pertussis ready to use PasteurSanté Canada (acellular) Limited Influenza, seasonal AGRIFLU Liquid:Seqirus Health Canada - ready to use Vaccines Santé Canada LimitedPneumococcal Prevenar 13 Liquid: Pfizer European (conjugate) Multidoseready to use Medicines Agency Vial Typhoid Typbar-TVC Liquid: BharatCentral Drugs (Conjugate) ready to use Biotech Standard ControlInternational Organization Limited Polio Vaccine - Oral PoliomyelitisLiquid: Beijing National Medical (OPV) Bivalent Types Vaccine ready touse Bio- Products 1 and 3 (live, oral Institute Administrationattenuated, Biological human Products Diploid Co., Ltd Cell), type 1 and3 Japanese JEEV ® Liquid: Biological Central Drugs Encephalitis ready touse E. Limited Standard Control Vaccine Organization (Inactivated) (3 μgPediatric) Rotavirus (live, ROTASIIL Lyophilized Serum Central Drugsattenuated) active Institute of Standard Control component India Pvt.Organization to be Ltd. reconstituted with excipient diluent before usePolio Vaccine - Poliomyelitis Liquid: Serum Central Drugs Inactivated(IPV) Vaccine ready to use Institute of Standard Control (Inactivated)India Pvt. Organization Ltd. Polio Vaccine - Poliomyelitis Liquid: SerumCentral Drugs Inactivated (IPV) Vaccine ready to use Institute ofStandard Control (Inactivated) India Pvt. Organization Ltd. Influenza,seasonal GCFLU Liquid: Green Ministry of Food Quadrivalent Quadrivalentready to use Cross and Drug Safety Multi inj. Corporation Influenza,seasonal Serinflu Liquid: Abbott Medicines ready to use BiologicalsEvaluation Board BV (MEB) Polio Vaccine - ShanIPV Liquid: ShanthaCentral Drugs Inactivated (IPV) ready to use Biotechnics StandardControl Private Organization Limited (A Sanofi Company) Polio Vaccine -Oral Bivalent Liquid: Panacea Central Drugs (OPV) Bivalent Types OPVType 1 ready to use Biotec Ltd. Standard Control 1 and 3 and 3Organization Poliomyelitis Vaccine, Live (Oral) cholera: inactivatedEuvichol- Liquid: EuBiologics Ministry of Food oral Plus ready to useCo., Ltd. and Drug Safety Polio Vaccine - Oral BIOPOLIO Liquid: BharatCentral Drugs (OPV) Bivalent Types B1/3 ready to use Biotech StandardControl 1 and 3 International Organization Limited BCG BCG FreezeLyophilized Japan BCG Chiba Local Dried active Laboratory GovernmentGlutamate component vaccine to be reconstituted with excipient diluentbefore use Pneumococcal Synflorix Liquid: GlaxoSmithKline European(conjugate) ready to use Biologicals Medicines Agency SA Rotavirus(live, Rotavac Liquid: Bharat Central Drugs attenuated) ready to useBiotech Standard Control International Organization Limited Hepatitis A(Human HEALIVE Liquid: Sinovac National Medical Diploid Cell), ready touse Biotech Products Inactivated Co. Ltd Administration (Paediatric)Typhoid Typbar-TVC Liquid: Bharat Central Drugs (Conjugate) ready to useBiotech Standard Control International Organization Limited Rotavirus(live, ROTASIIL Lyophilized Serum Central Drugs attenuated) activeInstitute of Standard Control component India Pvt. Organization to beLtd. reconstituted with excipient diluent before use Japanese JEEV ®Liquid: Biological Central Drugs Encephalitis ready to use E. LimitedStandard Control Vaccine Organization (Inactivated) 6 μg Japanese JEEV ®Liquid: Biological Central Drugs Encephalitis ready to use E. LimitedStandard Control Vaccine Organization (Inactivated) (3 μg Pediatric)SARS-CoV-2 PFIZER- Liquid: Pfizer- CEBR/FDA BIONTECH ready to useBIONTECH COVID-19 VACCINE- bnt162b2 SARS-CoV-2 Moderna Liquid: ModernaCEBR/FDA COVID-19 ready to use Inc SARS-CoV-2 COVID-19 Liquid: AstraEuropean Vaccine ready to use Zeneca Medicines Agency (ChAdOx1-S[recombinant]) SARS-CoV-2 Janssen Liquid: Johnson & CEBR/FDA COVID-19ready to use Johnson Vaccine SARS-CoV-2 CoronaVac, Liquid: SinovacNational Medical COVID-19 ready to use Products Vaccine Administration(Vero Cell), Inactivated

In various embodiments, the epitope is released following proteolyticcleavage of the peptide from the IRC. After proteolytic cleavage of thepeptide from the IRC, the epitope binds to an MHC, optionally an MHCclass I, molecule. The MHC molecule is in some embodiments from theHLA-A, B, and/or HLA C families. The specific epitope that binds to theMHC class I molecule is any of those recited in Table 2 or Table 3 orfound elsewhere in the art. The MHC class I molecule itself is, in someembodiments, one or more of the following non-limiting examples:HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, HLA-A*201, HLA-A*020101,HLA-A*0203, HLA-A*0206, HLA-A2, HLA-A2.1, or HLA-A*02.

In an aspect the described methods, uses, and compositions, the epitopeis about 8 amino acid to about 50 amino acids in length, or about 8amino acid to about 45 amino acids in length, or about 8 amino acid toabout 40 amino acids in length, about 8 amino acid to about 35 aminoacids in length, or about 8 amino acid to about 30 amino acids inlength, about 8 amino acid to about 25 amino acids in length, about 8amino acid to about 20 amino acids in length, or is about 8 amino acidto about 15 amino acids in length. In an aspect of the invention thepeptide is about 13 amino acid to about 50 amino acids in length, orabout 13 amino acid to about 45 amino acids in length, or about 13 aminoacid to about 40 amino acids in length, about 13 amino acid to about 35amino acids in length, or about 13 amino acid to about 30 amino acids inlength, about 13 amino acid to about 25 amino acids in length, about 13amino acid to about 20 amino acids in length, or is about 13 amino acidto about 15 amino acids in length. In some embodiments, the CD8+ T cellepitope is, e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or about 17amino acids in length.

Cleavage Sequence. In various embodiments, one or more protease cleavagesequences are incorporated into the IRC that, upon cleavage, allows thepeptide to be released from the IRC so that the peptide then is free tobind to the MHC on the tumor or cancer cell surface. In variousembodiments, the IRC must escape the endosome, disassemble, and releasetheir therapeutic cargo to the cytosol in a functional form. In variousembodiments the IRC and/or peptide of the IRC is susceptible to cleavageby a proteolytic enzyme within the tumor microenvironment, i.e., in thenearby interstitial space surrounding tumors or tumor cells, and theposition of the target cleavage sequence in the IRC or peptide is suchthat the cleavage of the target site releases all or a portion of thepeptide comprising the CD8+ T cell epitope from the IRC, which then isfree to bind to, and/or form a complex with, an MHC molecule expressedon the surface of the tumor cell in the subject. Pharmaceuticallyeffective, or therapeutic amounts of IRC required to achieve this endgoal are determined by the skilled artisan by known clinical methodsutilizing in vitro cell culture techniques, animal model studies, andsmall scale to large scale human clinical trials. It will be appreciatedthat the amount of IRC administered to the subject in need thereof inthe described methods and uses herein will depend on, e.g., thecharacteristics of the subject, e.g., age, weight, gender, and/ormedical condition/history, genetic makeup, and other factors pertinentto the subject or class of subjects, and that the characteristics of thetumor, e.g., type, volume, and developmental status will also be takeninto account when designing the dosage range finding clinical studies.

The proteolytic cleavage sequence is in some embodiments recognized byany protease present in, on, around, or nearby a tumor cell. At leastabout 569 known proteases have been described. (See, Lopez-Otin, et al.,Nature Reviews Cancer, 7(10):800-808, 2007). All human proteolyticenzymes identified to date are classifiable into five catalytic classes:metalloproteinases, serine, threonine, cysteine, and aspartic proteases.A non-limiting list of potential proteases is demonstrated in Table 4,which is a table summarizing exemplars of the most well-studiedproteases distributed into the five noted classes. (See Choi, Ki Younget al., “Protease-activated drug development,” Theranostics,(2)2:156-78, 2012). Several of these proteases have been found to beover-expressed in cancer cells relative to healthy cells.

In various embodiments, the proteolytic cleavage sequence is recognizedby the protease furin, a matrix metalloproteinase (MMP), of whichseveral different members are identified, e.g., MMP, 1, 2, 3, 7, 8, 9,11, 13, 14, or 19, an ADAM (a disintegrin and metalloproteinase), e.g.,ADAMS 8, 9, 10, 15, 17, or 28, a cathepsin, e.g., cathepsin D, G, H, orN. Also contemplated herein are the proteases elastase, proteinase-3,azurocidin, and ADAMTS-1. In various embodiments, the cleavage sequenceis recognized by any one or more of the aforementioned proteases, and ina certain embodiment the sequence is recognized by a human furinprotease. In various embodiments, the cleavage sequence comprises atleast about 4 amino acid residues, at least about three of which arearginine residues. In various embodiments, the cleavage sequencecomprises at least 4 amino acid residues, at least three of which arearginine residues and one of which is either a lysine residue or anarginine residue. In various embodiments, the cleavage sequence isR—X-R/K-R (SEQ ID NO: 89). In various embodiments, the cleavage sequencecomprises additional residues. In various embodiments, the cleavagesequence further comprises about 1, 2, 3, 4, 5, 6, 7, 8, or about 9additional arginine residues. It is known that arginines are positivelycharged and it has been discovered that a longer chain of positivecharged arginine residues will bring the peptides closer to the surfaceof the capsid backbone which is more negatively charged.

TABLE 4 Proteases and cancers associated with overexpressed proteasesFamily Protease Location Cancer Ref. Other Diseases Ref. CysteineGeneral Intracellular, Most Table in Cathepsins lysosomes [121]Cathepsin K Extracellular, Breast [178] Artherosclerosis, [179-182] boneosteoporosis Cathepsin B Extracellular Breast, cervix, colon, [31, 38,81, and colorectal, gastric, head and 183-196] pericellular neck, liver,lung, melanoma, under ovarian, pancreatic, prostate, pathologicalthyroid conditions Aspartic Cathepsin L Breast, colorectal  [28] AD[197] Cathepsins Cathepsin E Endosomal Cervical, gastric, lung, [51-55]structures, ER, pancreas adenocarcinomas Golgi Cathepsin D LysosomeBreast, colorectal, ovarian [47-49, Atherosclerosis [121] 198-200]General Intracellular, Most Table in secreted [15, 58] Kallikreins hK1Hypertension,  [24] (hK) inflammation PSA (hK 3) Prostate, ovarian[201-202] hK10 Colon, ovarian, pancreatic, [203-206] head and neck hK15Ovarian, prostate [207-208] Serine uPA, uPAR Membrane, Cervical,colorectal, gastric, [86, 116, Proteases Pericellular prostate 209-210]Caspases Intracellular Neurodegenerative  [82] disorders GeneralExtracellular Most Table in [211] MMPs MMP-1, -8, -13 Breast [85,102-104, Artherosclerosis, RA [213-214] 211-212] MMP-2, -9 Breast,colorectal, lung, [91-94] Bronchiectasis, chronic [87, 113- malignantgliomas, ovarian [95-98] asthma, COPD, cystic 117] fibrosis, HIVassociated dementia, hypertension, stroke MMP-14 Membrane Breast [212]ADAM Extracellular AD [105, 107, 112] *Abbreviations: AD: Alzheimer'sdisease; ADAM: a disintegrin and metalloproteinase domain protease;COPD: chronic obstructive pulmonary disease; ER: endoplasmic reticulum;RA: rheumatoid arthritis

In various embodiments, the peptide is bound to the capsid backbone, asdescribed in more detail below. There are multiple known means by whichthe peptide is able to be associated with, or bound to, the capsidbackbone. In various embodiments of the present disclosure the cleavagesequence is chemically conjugated by way of a maleimide linkage or anamide linkage (discussed below). The peptide is generally linked to anyresidue on the capsid backbone; however, disulfide linkages, maleimidelinkages, and amide linkages are formed by conjugating the peptide tocysteine, lysine, or arginine residues of the mutant L1 proteins thatcomprise the capsid backbones.

In various embodiments the peptide comprises at least one proteasecleavage sequence. In some embodiments, the protease cleavage sequenceis any sequence capable of being preferentially cleaved by or near atumor cell. The insertion of this cleavage sequence into the peptideallows the protein to remain attached to the capsid backbone carrieruntil the IRC enters the tumor microenvironment. By taking advantage ofthe elevated activities of particular proteases in cancer tissues ortumor microenvironments, the peptide is to a large extent not releasedfrom the capsid backbone and able to actively coat MHC receptors untilthe peptide enters the tumor microenvironment. Several proteases areknown in the art to be active in the tumor microenvironment. Forexample, several metallo-, cysteine and serine proteases are known. Fromthe standpoint of cancer therapy, an additional attraction is thatbecause the proteases responsible for prodrug cleavage may come not justfrom cancer cells but also from the stromal components of tumors,release of the active drug direction into the tumor microenvironmentdoes not depend on a target expressed only by the cancer cells. Instead,it is the entire tumor ecosystem that represents the target.

Methods of Attaching Peptides to the L1 Protein

The capsid backbones described herein are in some embodiments firstfunctionalized to deliver an epitope containing on one or more peptidesassociated with the capsid backbone to the target cells, therebylabeling the tumor or cancer cells for destruction. In variousembodiments, peptides are conjugated to the capsid backbone throughcysteine residues on the capsid protein. Such cysteine molecule arepresented naturally, or by mutation, on the surface of the capsidbackbone. In various embodiments, the capsid backbone is subjected toreducing conditions sufficient to reduce the sulfhydryl groups ofcysteine residues on the surface of the capsid backbone whilemaintaining the capsid-like icosahedron structures of the capsidbackbone. Because of its free sulfhydryl group, cysteine will readilyand spontaneously form disulfide bonds with other sulfhydryl-containingligands under oxidative conditions. Alternatively, a series of compoundsare known to add a maleimide moiety to receptive substrates that readilyand irreversibly form thioester linkages with cysteine residues at a pHbetween about 6.5 and about 7.5. Thus, in one embodiment, the peptide isassociated with the capsid backbone via a maleimide linkage.

In various embodiments, the peptide is conjugated to a lysine residue onthe capsid backbone. Lysine residues are easily modified because oftheir primary amine moiety. Using reactions termed n-hydroxysuccinimide(NHS) ester reactions (because NHS is released as prat of the reaction),amide bonds are formed at surface-exposed lysine residues on the capsidbackbone. The NHS reaction occurs spontaneously between about pH 7.2 andabout pH 9.

In various embodiments, the peptide is conjugated to an aspartate orglutamate residue. Unlike chemical coupling strategies involvingcysteine and lysine groups, chemically coupling to aspartate orglutamate residues requires multiple steps. First, the carboxylic acidof the aspartate or glutamate is activated using1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), orsimilar chemical cross-linking reagent. Once activated, this adduct willreact with NHS to form an NHS ester. The NHS ester is then reacted witha ligand with an exposed primary amine to from a stable amide bond.

In various embodiments, the capsid backbone comprises a region ofnegatively charged amino acids on a surface-exposed area that is capableof binding to the peptide comprising a region of positively chargedamino acids. In various embodiments, the region of negatively chargedamino acids is flanked, on one or on both sides, by one or more cysteineresidues, referred to as polyanionic: cysteine or more specifically,polyglutamic acid:cysteine or polyaspartic acid:cysteine. In such cases,the conjugation of the capsid backbone and the peptide would result fromnon-covalent binding between the complementary amino acid charges of thecapsid backbone and the peptide and a disulfide bond between thecysteines. In various embodiments, the cysteine(s) are one or more aminoacids away from the region of charged amino acids such that anysecondary/tertiary structure would bring the charged amino acid regionin close proximity to the cysteine(s). In various embodiments, thepeptide comprises at least one peptide and a polyionic:cysteine forattaching the peptide to the capsid backbone comprising a complementarypolyionic:cysteine sequence and an enzyme cleavage site positionedbetween the terminal cysteine and the CD8+ T cell epitope. In variousembodiments, the peptide comprises, a terminal cysteine, at least onepeptide, and an enzyme cleavage sequence positioned between the terminalcysteine and the peptide(s).

Negatively charged amino acids that are useful in producing thedescribed IRC include, e.g., glutamic acid and aspartic acid. Theseamino acids are used singly in some embodiments, e.g., polyglutamicacid, or in combination. In a specific embodiment, the negativelycharged region comprises glutamic acid. The number of negatively chargedamino acids can vary and can include about 4 to about 20 amino acids,about 6 to about 18 amino acids, or about 8 to about 16 amino acids, andthe like. In a specific embodiment, the negatively charged regioncomprises about 8 negatively charged amino acids. In a more specificembodiment, the negatively charged region comprises EEEEEEEEC (E8C, SEQID NO: 130). In another embodiment, the negatively charged regioncomprises CEEEEEEEEC (SEQ ID NO: 131). Methods for conjugating peptidesto a capsid backbones via disulfide bonding are known. For instance, thepresence of a polyarginine-cysteine moiety on the peptide allows dockingof the peptide to the polyanionic site (EEEEEEEEC, E8C, SEQ ID NO: 130)present in the various loops of the capsid backbone. Covalentcross-linking between the two cysteine residues should render thisassociation irreversible under oxidizing conditions. For the conjugationreactions, purified capsid backbones are dialyzed in conjugation buffer(20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5% glycerol, 0.5 mM CaCl₂) andthen the peptide and the oxidizing reagents are added, allowing thereaction to proceed for 16 hrs at 4° C. At the end of the incubation,the reaction mixtures are applied to a size-exclusion column (such asSEPHADEX® G-100, Pharmacia, New Jersey, US, volume 20 ml, flow rate 1ml/min, 10 mM Tris/HCl (pH 7.4), 150 mM NaCl, 0.5 mM CaCl₂)) to removeunconjugated peptide and exchange buffer. IRCs that elute in the voidvolume are identified by the presence of the L1 protein on SDS-PAGE. Theconjugated capsid backbones (IRC) are than optionally analyzed byelectron microscopy.

In various embodiments, the peptide is genetically fused to the L1protein. In various embodiments, the peptide is either covalently ornon-covalently linked to the capsid backbone. Rather than attaching thepeptide to the capsid backbone via, e.g., binding of negatively andpositively charged amino acids, or via maleimide based conjugation, anucleic acid sequence encoding the peptide is inserted in someembodiments into the nucleic acid encoding the L1 protein such that uponexpression a peptide is produced that is inserted into a loop of thecapsid protein and displayed on the surface of the capsid backbone.

In various embodiments, non-natural amino acids are used to conjugatethe peptide to the capsid backbone. Beyond the 20 natural amino acids,many non-natural amino acids have been used for site-specific proteinconjugation reactions. For example, an azidohomoalanine (AHA) or ap-amino-phenylalanine (pAF) may be incorporated into the capsid backbonecoat protein for conjugation. These amino acids are incorporated intoproteins in two ways: global methionine replacement and amber stop codonsuppression. Because AHA is very similar to methionine, AHA will beincorporated at each AUG codon if the methionine supply is ratelimiting, this is termed global methionine replacement. Bacteriaauxotrophic for methionine or cell-free protein synthesis can be used tolimit-methionine availability. Amber stop codon suppression willincorporate pAF. Amber stop codon suppression uses nonnative synthetasesand tRNAs that do not react with the natural amino acids to incorporatethe non-natural amino acid at the amber stop codon UAG. AHA, displayingan azide, will participate in in copper(I)-catalyzed azide-alkynecycloaddition (“click” reaction) and form covalent triazole rings withalkyne-containing ligands.

In various embodiments, the IRC comprises, at least one-tenth of the L1proteins display a peptide. In various embodiments, at least one-fifthof the L1 proteins display a peptide. In various embodiments, about halfof the L1 proteins display a peptide. In various embodiments, abouttwo-thirds of the L1 proteins display a recall peptide. In variousembodiments, nearly all of the L1 proteins display a peptide.

IRCs and Uses Thereof in Clinical Therapies

In various embodiments, the capsid backbone binds preferentially totumor cells. The capsid backbones' tumor preference originates, in someembodiments, from several sources such as the capsid backbone's charge(positive or negative), shape and size (different aspect ratio filamentsand diameter spheres), shielding (self-proteins/peptides and polymers ofvarious sizes and densities), and targeting (ligands for receptors orenvironmental factors displayed on different linkers at variousdensities).

In terms of charge, in various embodiments, the capsid backbone containsa positive surface charge. Positively charged capsid backbones have beenshown in some studies to remain longer in circulation when injected intoa subject. Due to the abundant presence of proteoglycan in cellmembranes that confer a negative charge to cell membranes, and collagenwithin the tumor interstitial space conferring a positive charge,positively charged IRCs are more likely to possess enhanced binding tomammalian cells as compared with non-charged or negatively charged IRCs,and therefor are better able to avoid aggregation and as a result, areable to better penetrate tumor tissue. Some examples demonstrating thesecharge-based effects include polyarginine-decorated cowpea mosaic virus(CPMV) found to be taken up eight times more efficiently than nativeCPMV in a human cervical cancer. (Wen et al. Chem. Soc. Rev.,45(15):4074-4126, 2016).

With regards to shape, the shape and flexibility of the capsid backbonein some instances plays an additional functional role in the ability ofcapsid backbones to diffuse throughout a tumor. A comparison between thediffusion profiles of a spherical and rod-shaped particle was performedwith CPMV and TMV using a spheroid model. It was shown in this studythat the CPMV (spherical) experienced a steady diffusion profile, butthe TMV (rod shaped) exhibited a two-phase diffusion behavior thatentailed an extremely rapid early loading phase that could be attributedto its movement axially, like a needle. (Wen et al., Chem. Soc. Rev.,45(15):4074-41:26, 2016). Some other advantageous properties that areconferred by elongated particles include better margination toward thevessel wall and stronger adherence due to greater surface area forinteraction, which not only have implications for tumor homing but alsofor enhanced targeting of cardiovascular disease.

Besides passive tumor homing properties, natural interactions of viruseswith certain cells can also be exploited. CPMV in particular exhibitsunique specificity in interacting with surface vimentin, which is foundon endothelial, cancer, and inflammatory cells. (Wen et al., Chem. Soc.Rev., 45(15):4074-4126, 2016). The native affinity of CPMV for surfacevimentin allows for high-resolution imaging of microvasculature up to500 μm in depth, which cannot be achieved through the use of othernanoparticles, as they tend to aggregate and block the vasculature. Thisinteraction can be utilized for a range of applications, such asdelivery to a panel of cancer cells including cervical, breast, andcolon cancer cell lines, delineation of atherosclerotic lesions, andintravital imaging of tumor vasculature and angiogenesis. Anotherexample of an existing endogenous association is canine parvovirus (CPV)with transferrin receptor (TfR), an important receptor for irontransport into cells and highly upregulated by numerous cancer celllines. Even after dye labelling, CPV retains its specificity for TfR andwas shown to bind to receptors found on HeLa cervical cancer cells,HT-29 colon cancer cells, and MDA-MB-231 breast cancer cells. (Wen etal., Chem. Soc. Rev., 45(15):4074-4126, 2016).

In various embodiments, the capsid backbone targets a protein expressedpreferentially on the tumor cell surface in the subject. Such proteinsare typically overexpressed on the surface of tumor cells, but some ifnot all, are also found in the blood, i.e., serum. Non-limiting examplesof such surface markers include: CEA (carcinoembryonic antigen),E-cadherin, EMA (epithelial membrane antigen; aka MUC-1), vimentin,fibronectin, Her2/neu (human epidermal growth factor receptor type 2,also called Erb b2), αvβ3 integrin, EpCAM (epithelial cell adhesionmolecule), FR-α (folate receptor-alpha), PAR (urokinase-type plasminogenactivator receptor), and transferrin receptor (over expressed in tumorcells).

Peptides are often used to label cancerous cells based on recognition oftheir transmembrane proteins. The most commonly used peptide isarginylglycylaspartic acid (RGD), which is composed of L-arginine,glycine, and L-aspartic acid. RGD was first isolated from thecell-binding domain of fibronectin, a glycoprotein that binds tointegrins, and is involved in cell-cell and cell-extracellular matrix(ECM) attachment and signaling by binding collagen, fibrin, andproteoglycans. RGD peptides have the highest affinity for a type of cellsurface integrins, αvβ which are highly expressed in tumoral endothelialcells, but not in normal endothelial cells. In various embodiments sucha peptide sequence is incorporated into the IRC.

Methods of treating cancers in a subject in need thereof byadministering an IRC to patient in need thereof, and related uses of thedescribed IRC compositions, are described herein. The methods describedherein comprise, for instance, administering the IRCs described hereinto a subject in need thereof in an amount sufficient to inhibit tumorgrowth, progression or metastasis, i.e., a therapeutic amount or dose.In various embodiments, the IRC is administered to a subject in needthereof in amount sufficient to stimulate cytokine production and/orcellular immunity, particularly innate immunity, including stimulationof the cytotoxic activity of macrophages and natural killer cells. Invarious embodiments described herein, a subject in need thereof is asubject who has been previously treated for a tumor and is currentlydeemed cancer-free or disease free in accordance with medical standards.

Briefly, various understood aspects of what is believed to be themechanism of action of the described IRCs are described and supported bythe examples, below. The IRC first bind to a tumor cell, in someembodiments the binding is specific. (See, Example 9, FIGS. 18A and18B). The peptide epitope on the IRC is then proteolytically cleaved byfurin, in some embodiments, or by any other resident protease nearby thetumor cell, which is over-expressed in the tumor microenvironment. Thisin turn leads to release of the peptide from the IRC and the loading, orbinding, of the peptide by an MHC molecule expressed on the surface ofthe tumor cell (“epitope coating”). (See, Examples 10 and 16, and FIGS.19, 21, 22, and 34). The epitope-coated tumor cell is then recognized asa pathogen-infected cell by one or more T-cells responsive to thespecific peptide bound in the MHC molecules, and pre-existing CD8 Tcells, yielding a triggered immune redirection response. (See, Examples11 and 12). That is, this recognition event leads to triggering oractivation of the subject's preexisting immune memory against pathogensand childhood vaccines against the tumor, leading to the attacking anddestroying of the subject's tumor cells.

Destruction of tumor cells can result in components of the preexistingimmune response being exposed to cancer cell antigens. Thus, antigensreleased from the killed tumor cells will initiate a further immuneresponse to recruit additional tumor-specific CD8 T cells, or a “secondwave” of T cells that then proceed to attack additional tumor cells inthe area. This can result in elicitation of an endogenous immuneresponse against the cancer cell antigens (referred in some instances to“epitope spreading”) and leads to anti-tumor immune memory.

Thus, the methods and uses disclosed herein are methods of treatingcancer in an subject in need thereof that occurs through utilizing, orthe re-orienting of, the subject's own preexisting adaptive memoryimmune system to attack cancer cells. The methods and uses describedherein make use of the fact that subjects, in some instances, possesspreexisting immune responses that were not originally elicited inresponse to a cancer, but that were elicited instead by routinevaccination or via natural infection by a parasite or pathogen. Becausethe cancer cells would not normally express such epitopes that elicitpreexisting immune responses, it would not be expected that such animmune response would not normally, without exogeneous intervention, becapable of attacking any cancer cell. However, by way of the presentmethods and uses described herein, such preexisting immune responses arereadily recruited to attack, kill, and clear a cancer in a subject. Thisrecruitment or repurposing effect is therefore achieved by way of thepresent IRC compositions since these IRC, upon injection or other meansof delivery into the subject, introduce into or onto the surface of thecancer one or more epitopes known to be recognized by the preexistingimmune response in the subject, resulting in cells of the immuneresponse attacking antigen-displaying cancer cells.

Thus, without wishing to be bound by any specific theory, the methods,uses, and compositions described herein act by recruiting a preexistingimmune response in a subject to the site of a cancer, such that thepreexisting immune response attacks and kills the cancer cells. Thus,there are generally four or five steps involved in the describedmethods, including: 1) binding IRC to the tumor cells, 2) cleavage ofthe epitope from the IRC, 3) MHC binding of the epitopes for display onthe tumor cell surface, 4) recognition of the loaded MHC by thesubject's pre-existing recalled immunity against the epitope, andoptionally 5) triggering of a second wave and longer-term anti-tumoralimmunity thereafter.

Data obtained from cell culture assays and animal studies are often usedin formulating a range of dosages for use in humans. The dosages of suchcompositions lie preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage varieswithin this range depending on the dosage form employed and the route ofadministration utilized. For any composition used in the methodsdescribed herein, the therapeutically effective dose is capable of beingestimated initially from cell culture assays. A dose is formulated inanimal models to achieve a circulating plasma concentration range thatincludes the IC₅₀ (the concentration of the test composition thatachieves a half-maximal inhibition of symptoms) as determined in cellculture. Such information is then used to accurately determine usefuldoses in humans. Levels in plasma are measured, for example, by highperformance liquid chromatography.

In many instances, it will be desirable to have multiple administrationsof the IRC-containing compositions, usually at most, at least, or notexceeding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or moredoses including all ranges therebetween. The administrations willnormally be at 1, 2, 3, 4, 5, 6, to 5, 6, 7, 8, 9, 10, 11, to 12week/month/year intervals, including all values and ranges therebetween, more usually from three- to five-week intervals.

In various embodiments, a method is provided for stimulating thecytotoxic activity of macrophages and natural killer (NK) cells byadministering to a subject in need thereof an effective amount of an IRCdescribed herein. The macrophages and natural killer cells are in someinstances those that are present in the tumor microenvironment. In oneaspect, the IRCs are administered to the subject in an amount effectiveto stimulate the cytotoxic activity of macrophages and natural killercells already present in the tumor microenvironment. In various otherembodiments, the IRCs are administered to the subject in an amounteffective to attract macrophages and natural killer cells to the tumormicroenvironment. In various embodiments, the IRCs are administered tothe subject in an amount effective to bind sufficient numbers ofantibodies to the peptide or IRC capsid itself to attract and stimulatemacrophages, neutrophils and natural killer cells.

In various embodiments, methods and uses are provided for redirectingthe cytotoxic activity of an existing memory CD8+ T cell to a tumor cellor tumor microenvironment by administering to a subject in need thereofan effective amount of the IRC described herein. Preferably, the T cellepitope of the peptide of the IRC is from a pathogen for which thesubject has been vaccinated or from a pathogen that has previouslyinfected the subject and the subject has memory CD8+ T cells thatrecognize the T cell epitope in complex with an MHC class I molecule onthe tumor cells. In an aspect described herein, the effective ortherapeutic amount of the IRC compositions described herein is an amountsufficient to attract the memory CD8+ T cell to the tumormicroenvironment. In another alternative aspect, the effective amount ofthe IRC is an amount sufficient to stimulate the memory CD8+ T cellpresent in the tumor microenvironment.

In various embodiments, the tumor is a small lung cell cancer,hepatocellular carcinoma, liver cancer, hepatocellular carcinoma,melanoma, metastatic melanoma, adrenal cancer, anal cancer, aplasticanemia, bile duct cancer, bladder cancer, bone cancer, brain/CNS cancer,breast cancer, cancer of unknown primary origin, Castleman disease,cervical cancer, colon/rectum cancer, endometrial cancer, esophaguscancer, Ewing family of tumors, eye cancer, gallbladder cancer,gastrointestinal carcinoid tumors, gastrointestinal stromal tumor(gist), gestational trophoblastic disease, Hodgkin disease, Kaposisarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia,liver cancer, lung cancer, lymphoma, malignant mesothelioma, multiplemyeloma, myelodysplastic syndrome, nasal cavity and paranasal sinuscancer, nasopharyngeal cancer. neuroblastoma, oral cavity andoropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer,penile cancer, pituitary tumors, prostate cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, stomachcancer, testicular cancer, thymus cancer, thyroid cancer, uterinesarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia,Wilms tumor, non-Hodgkin lymphoma, Hodgkin lymphoma, Burkitt's lymphoma,lymphoblastic lymphomas, mantle cell lymphoma (MCL), multiple myeloma(MM), small lymphocytic lymphoma (SLL), splenic marginal zone lymphoma,marginal zone lymphoma (extra-nodal or nodal), mixed cell type diffuseaggressive lymphomas of adults, large cell type diffuse aggressivelymphomas of adults, large cell immunoblastic diffuse aggressivelymphomas of adults, small non-cleaved cell diffuse aggressive lymphomasof adults, or follicular lymphoma, head and neck cancer, endometrial oruterine carcinoma, non-small cell lung cancer, osteosarcoma,glioblastoma, or metastatic cancer. In a preferred embodiment, thecancer is a breast cancer, a cervical cancer, an ovarian cancer, apancreatic cancer or melanoma,

The term “cancer” as used herein refers to proliferative diseases, suchas lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung(NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrine systemcancer of the thyroid gland, cancer of the parathyroid gland, cancer ofthe adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancerof the penis, prostate cancer, cancer of the bladder, cancer of thekidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis,mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of thecentral nervous system (CNS), spinal axis tumors, brain stem glioma,glio-blastoma multiforme, astrocytomas, schwanomas, ependymonas,medulloblastomas, meningiomas, squamous cell carcinomas, pituitaryadenoma and Ewing's sarcoma, including refractory versions of any of theabove cancers, or a combination of one or more of the above cancers.

An aspect described herein is a method for treating a cancer in asubject in need thereof by administering an IRC described herein to thesubject wherein the CD8+ epitope of the peptide is of a failedtherapeutic cancer vaccine against a viral-induced cancer, e.g., HPVcervical cancer, HPV+ oral cancer, EBV nasopharyngeal cancer (the“therapeutic vaccine”). The methods and uses described herein thereforecomprise determining whether the subject has been actively vaccinatedbut did not respond with an anti-tumor effect to the treatment. The IRCcomposition is then administering to the subject an effective amount ofan IRC of this invention wherein the CD8+ epitope of the peptide is ofthe antigenic determinant in the vaccine previously administered to thesubject that infected the subject.

Capsid backbones have inherent adjuvant properties. In some embodiments,the immunogenicity of the IRC compositions described herein are furtherenhanced by the combination with additional nonspecific stimulators ofthe immune response, known as adjuvants. Suitable adjuvants include allacceptable immunostimulatory compounds, such as, but not limited to,cytokines, toxins, or synthetic compositions such as alum.

Adjuvants include, but are not limited to, oil-in-water emulsions,water-in-oil emulsions, mineral salts, polynucleotides, and naturalsubstances. Specific adjuvants that may be used include IL-1, IL-2,IL-4, IL-7, IL-12, y-interferon, GM-CSF, BCG, aluminum salts, such asaluminum hydroxide or other aluminum compound, methylenedioxyphenyl(MDP) compounds, such as thur-MDP and nor-MOP, CGP (MTP-PE), lipid A,and monophosphoryl lipid A (MPL), or inactivated microbial agents. RIBI,which contains three components extracted from bacteria, MPL, trehalosedimycolate (TOM), and cell wall skeleton (CWS) in a 2% squalene/Tween 80emulsion. MHC antigens may even be used.

Various methods of achieving adjuvant affect for the IRC compositionsincludes use of agents such as aluminum hydroxide or phosphate (alum),commonly used as about 0.05 to about 0.1% solution in phosphate bufferedsaline, admixture with synthetic polymers of sugars (CARBOPOL®) used asan about 0.25% solution, aggregation of a protein in the composition byheat treatment with temperatures ranging between about 70° C. to about101° C. for a 30-second to 2-minute period, respectively. Aggregation byreactivating with pepsin-treated (Fab) antibodies to albumin; mixturewith bacterial cells, e.g., C. parvum, endotoxins or lipopolysaccharidecomponents of Gram-negative bacteria; emulsion in physiologicallyacceptable oil vehicles, e.g., mannide monooleate (Aracel ATM), oremulsion with a 20% solution of a perfluorocarbon (FLUOSOL-DA®) used asa block substitute may also be employed to produce an adjuvant effect. Atypical adjuvant is complete Freund's adjuvant (containing killedMycobacterium tuberculosis), incomplete Freund's adjuvants, and aluminumhydroxide.

For administration to humans, a variety of suitable adjuvants will beevident to a skilled worker. These include, e.g., Alum-MPL as adjuvant,or the comparable formulation, ASO4, which is used in the approved HPVvaccine CERVARIX®, AS03, AS02, MF59, montanide, saponin-based adjuvantssuch as GPI-0100, CpG-based adjuvants, or imiquimod. In embodiments ofthe invention, an adjuvant is physically coupled to the capsid backbone,or encapsulated by the capsid backbone, rather than simply mixed withthem. In addition to adjuvants, it may be desirable to co-administerbiologic response modifiers (BRM) to enhance immune responses. BRMs havebeen shown to upregulate T cell immunity or downregulate suppresser cellactivity. Such BRMs include, but are not limited to, Cimetidine (CIM;1200 mg/d) (Smith/Kline, PA, US); or low-dose Cyclophosphamide (CYP; 300mg/ml) (Johnson/Mead, NJ, US) and cytokines such as γ-interferon, IL-2,or IL-12 or genes encoding proteins involved in immune helper functions,such as B-7. In embodiments described herein, these genes areencapsulated by the capsid backbone to facilitate their delivery into asubject.

The preparation of compositions that contain polypeptide or peptidesequence(s) as active ingredients is generally well understood in theart. Typically, such compositions are prepared as injectables either asliquid solutions or suspensions: solid forms suitable for solution in orsuspension in liquid prior to injection may also be prepared. Thepreparation is in some instances emulsified. The active immunogenicingredient is in some embodiments mixed with excipients that arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof. In addition, if desired,the compositions may contain amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, or adjuvants thatenhance the effectiveness of the vaccines. In specific embodiments,vaccines are formulated with a combination of substances.

The compositions comprising the IRCs of the present disclosure areintended to be in a biologically-compatible form that is suitable foradministration in vivo to subjects. The pharmaceutical compositionsdescribed herein further comprise one or more optional pharmaceuticallyacceptable carriers. The term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government,e.g., the FDA, or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly, inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the capsid backbone is administered. Suchpharmaceutical carriers include, for example, sterile liquids, such aswater and oils, including those of petroleum, animal, vegetable orsynthetic origin, including but not limited to peanut oil, soybean oil,mineral oil, sesame oil and the like. Water is a carrier in someinstances when the pharmaceutical composition described herein isadministered orally. Saline and aqueous dextrose are carriers, forexample, when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions are employed, for instance, as liquid carriers for injectablesolutions. Suitable pharmaceutical excipients include, withoutlimitation, starch, glucose, lactose, sucrose, gelatin, malt, rice,flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc,sodium chloride, dried slim milk, glycerol, propylene, glycol, water,ethanol and the like. The pharmaceutical composition in some embodimentsoptionally contains minor amounts of wetting or emulsifying agents, orpH buffering agents.

The pharmaceutical compositions comprising the IRCs of the presentdisclosure take the form of, for example, solutions, suspensions,emulsions, tablets, pills, capsules, powders, sustained-releaseformulations, and the like. Oral formulation includes in someembodiments standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. In a specific embodiment, a pharmaceuticalcomposition comprises an effective amount of an IRC of the presentdisclosure together with a suitable amount of a pharmaceuticallyacceptable carrier so as to provide the form for proper administrationto the subject. The formulation should suit the mode of administration.

The pharmaceutical compositions of the present disclosure areadministered by any particular route of administration including, butnot limited to, intravenous, intramuscular, intraarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracerebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intraos seous, intrapelvic, intrapericardial,intraperitoneal, intrapleural, intraprostatic, intrapulmonary,intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial,intrathoracic, intrauterine, intravesical, bolus, oral, parenteral,subcutaneous, vaginal, rectal, buccal, sublingual, intranasal,iontophoretic means, or transdermal means. Most suitable routes areintravenous injection or oral administration. In particular embodiments,the compositions are administered at or near the target area, e.g.,intratumoral injection.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered, if necessary, and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, intratumoral, subcutaneous, and intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage could be dissolved inisotonic NaCl solution and either added to hypodermoclysis fluid orinjected at the proposed site of infusion. (See, for example,Remington's Pharmaceutical Sciences, 1990). Some variation in dosagenecessarily occurs depending on the condition of the subject. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject.

The IRC-containing compositions described herein, in some embodiments,are administered by inhalation. In certain embodiments a composition isadministered as an aerosol. As used herein the term “aerosol” or“aerosolized composition” refers to a suspension of solid or liquidparticles in a gas. These terms are used generally to refer to acomposition that has been vaporized, nebulized, or otherwise convertedfrom a solid or liquid form to an inhalable form including suspendedsolid or liquid drug particles. Such aerosols can be used to deliver acomposition via the respiratory system. As used herein, “respiratorysystem” refers to the system of organs in the body responsible for theintake of oxygen and the expiration of carbon dioxide. The systemgenerally includes all the air passages from the nose to the pulmonaryalveoli. In mammals it is generally considered to include the lungs,bronchi, bronchioles, trachea, nasal passages, and diaphragm. Forpurposes of the present disclosure, delivery of a composition to therespiratory system indicates that a drug is delivered to one or more ofthe air passages of the respiratory system, in particular to the lungs.

Additional formulations that are suitable for other modes ofadministration include suppositories (for anal or vaginal application)and, in some cases, oral formulations. For suppositories, traditionalbinders and carriers may include, for example, polyalkalene glycols ortriglycerides: such suppositories may be formed from mixtures containingthe active ingredient in the range of about 0.5% to about 10%,preferably about 1% to about 2%. Oral formulations include such normallyemployed excipients as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate and the like. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations, or powders and contain about 10% to about 95% of activeingredient, preferably about 25% to about 70%.

The IRC compositions described herein are, in some instances, formulatedinto a vaccine as neutral or salt forms. Pharmaceutically-acceptablesalts include the acid addition salts (formed with the free amino groupsof the peptide) and those that are formed with inorganic acids such as,for example, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed with thefree carboxyl groups may also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The pharmaceutical compositions of the present disclosure also include,in certain embodiments, an effective amount of an additional adjuvant.As noted herein, papillomavirus capsid backbones have adjuvantproperties. Suitable additional adjuvants include, but are not limitedto, Freund's complete or incomplete, mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, dinitrophenol, andpotentially useful human adjuvants such as Bacille Calmette-Guerin(BCG), Corynebacterium parvum, and non-toxic cholera toxin.

Under ordinary conditions of storage and use, the described IRCcompositions in some embodiments also contain a preservative to preventthe growth of microorganisms. In all cases the pharmaceutical form mustbe sterile and must be fluid to the extent that it may be easilyinjected. It also should be stable under the conditions of manufactureand storage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi.

The carrier is in some embodiments a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity ismaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersion,and by the use of surfactants. The prevention of the action ofmicroorganisms is brought about in some instances by incorporation ofvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions is achieved by the addition to the compositions of agentsdelaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the IRCs inthe required amount in the appropriate solvent with various ingredientsenumerated above, as required may be followed by filtered sterilization.Generally, dispersions are prepared by incorporating the varioussterilized active ingredients into a sterile vehicle which contains thebasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum-drying and freeze-drying techniques, which yield a powder of theactive ingredient, plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Different aspects of the present disclosure involve administering aneffective amount of a composition comprising the IRCs to a subject inneed thereof. In some embodiments of the present disclosure, an IRCcomprising a target peptide comprising a CD8+ T cell epitope isadministered to the patient to treat a tumor or prevent the recurrenceof such tumor. Such compositions will generally be dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.

Design of IRC for Specific Uses

In various embodiments, a method for providing an IRC to a subject inneed thereof is provided comprising: (1) measuring the preexistingimmunity in a subject, and (2) selecting the appropriate IRC foradministration of a subject in need. The appropriate IRC to administerto the subject will depend upon the patients T cell profile. Theappropriate IRC will be one that is capable of eliciting a T cellresponse that is at least twice the baseline total of CD8+ cells. Invarious embodiments, the appropriate IRC will be one that is capable ofeliciting a T cell response that is twice the baseline total of CD8+ ortotal CD8+CD69+ T cells. The goal is to choose the appropriate IRC basedon the subject's vaccination history or prior exposure to a pathogen.Determining which IRC is appropriate is, for example, achieved through:(1) subject interviews; (2) review of a subject's medical records;and/or (3) assessing the subject's T cell profile.

In various embodiments, more than one peptide is suitable for elicitingan immune response directed at a tumor. In various embodiments, an IRCcarrying either peptide or a mixture of both peptides will beappropriate. In various embodiments, more than one peptide is expressedand bound to the capsid backbone. In various embodiments, a singlepeptide will comprise more than one peptide. In various embodiments,multiple peptides comprising different peptides will be conjugated tothe capsid backbone. In various embodiments, the invention comprises apopulation of IRCs as described herein and a pharmaceutically acceptableexcipient. In various embodiments, the IRCs administered to the subjectare identical. In various embodiments, IRCs carrying differentpeptide(s) are administered to a subject.

Selection Based on Prior Vaccination. In various embodiments of themethods and uses described herein, contemplated is also a method ofselecting an appropriate IRC to administer to a subject in need thereof.In various embodiments this involves ascertaining if the subject hasbeen actively vaccinated against a given pathogen, e.g., a parasite, abacterium, or virus, e.g., measles or polio, and then selecting andadministering to the subject an IRC as disclosed herein wherein the CD8+T cell epitope of the peptide is from the pathogen against which thesubject has been immunized in the past. In various embodiments, asubject's vaccination history is obtained by reviewing the subject'smedical record. In various embodiments, a subject's vaccination historyis obtained by interviewing the subject.

Selection Based on Prior Infection. In various embodiments, the methodof selecting an appropriate IRC for administration to a subject in needthereof involves ascertaining if a subject has been previously infectedwith a given pathogen, e.g., a parasite, a bacterium, or virus, e.g.,measles or polio, and resolved the infection. In various embodiments,the subject is then administered an IRC comprising a peptide whichcomprises said pathogen for which the subject has been previouslyinfected.

One may ascertain if a subject has been infected with a particularpathogen by reviewing the subject's medical records or interviewing thesubject. Non-limiting examples of CD8+ T cell epitopes that bind toparticular MHC class I molecules are set forth in Table 1. The methodalso comprises, in certain embodiments, determining which MHC class Ideterminant(s) the subject's cells express and then administering an IRCdescribed herein wherein the CD8+ T cell epitope of the peptide is aCD8+ T cell epitope of the antigenic component of the pathogen in thevaccine or of the pathogen that previously infected the subject thatforms a complex with the subject's MHC class I determinant(s).

Measuring T cell Responses. In various embodiments, a subject's T cellprofile is also assessed in order to select an appropriate IRC usingvarious techniques known in the art. This profile is then used to guideselection of the appropriate IRC to administer to the subject. Suchtechniques include, for example, measuring interferon-γ levels, usingflow cytometry to isolate Ag-specific CD8+ T cells, and/or cytotoxicityassays. To measure interferon-γ (a marker of T cell activation),intracellular staining of isolated T cells. Alternatively, anenzyme-linked immunosorbent spot (ELISPOT) assay for interferon-γ may beconducted. This technique allows for a high throughput assessment of apatient's T cell profile. This method can potentially detect one in100,000-300,000 cells. Briefly, a monoclonal antibody for a specificcytokine is pre-coated onto a polyvinylidene difluoride (PVDF)-backedmicroplate. CD8+ T cells are pipetted into the wells along withdendritic cells and individual peptides and the microplate is placedinto a humidified 37° C. CO₂ incubator for a period ranging from 24 to48 h. During incubation, the immobilized antibody binds the cytokinesecreted from the cells. After washing a detection antibody specific forthe chosen analyte is added to the wells. Following the washes, enzymeconjugated to streptavidin is added and a substrate is added. A coloredprecipitate forms, according to the substrate utilized and appears asspot at the sites of cytokine secretion, with each individual spotrepresenting a single producing cell.

In various embodiments, provided are methods of determining theappropriate IRC to administer to a subject in need thereof, by assessingthe subject's T cell profile, comprising: (1) collecting PBMCs fromsubject (pre-vaccination sample), (2) preparing enzyme-linked immuneabsorbent spot (ELISPot) plates by coating with anti-IFN-γ antibody(incubate overnight), (3) incubating PBMCs with one of the pool ofpeptides of interest, i.e., the peptides expected to elicit a T cellresponse (incubate for 1-2 days), (4) washing the plates, adding abiotinylated secondary antibody (incubating for a few hours), (5)washing the plates, adding avidin conjugated horseradish peroxidase andincubating, (6) washing plates, adding aminoethyl carbazole (AEC) for afew minutes, (7) stopping the reaction (by adding water), and (8)visualizing on an ELISPot reader. The disclosed methods detect up to onein 100,000 to 300,000 cells. A two-fold increase in the frequency ofantigen-specific T cells should be considered as a signal.

In various embodiments T cell proliferation is measured by 3H(tritiated)-thymidine. Such methods are sensitive and can be used forhigh throughput assays. Such techniques include, for instance,carboxyfluorescein succinimidyl ester (CFSE) and Ki64 intracellularstaining.

Selecting Peptides based on Tropism. It is known in the art that someviruses display a tropism for particular type of tissue. For example:viruses that display a tropism for brain tissue include withoutlimitation, JC virus, measles, LCM virus, arbovirus and rabies; virusesthat display a tropism for eye tissue include without limitation herpessimplex virus, adenovirus, and cytomegalovirus; viruses that display atropism for nasal tissue include without limitation, rhinoviruses,parainfluenza viruses, and respiratory syncytial virus; viruses thatdisplay a tropism for oral tissue, e.g., oral mucosa, gingiva, salivaryglands, pharynx, include without limitation, herpes simplex virus type Iand type II, mumps virus, Epstein Barr virus, and cytomegalovirus;viruses that display a tropism for lung tissue include withoutlimitation, influenza virus type A and type B, parainfluenza virus,respiratory syncytial virus, adenovirus, and SARS coronavirus; virusesthat display a tropism for nerve tissue, e.g., the spinal cord, includewithout limitation poliovirus and HTLV-1; viruses that display a tropismfor heart tissue, include without limitation, Coxsackie B virus; virusesthat display a tropism for liver tissue, include without limitation,hepatitis viruses types A, B, and C; viruses that display a tropism forgastrointestinal tissue, e.g., stomach, and large and small intestine,include without limitation, adenovirus, rotavirus, norovirus,astrovirus, and coronavirus; viruses that display a tropism forpancreatic tissue, include without limitation, coxsackie B virus;viruses that display a tropism for skin tissue, include withoutlimitation, varicella zoster virus, herpes simplex virus 6, smallpoxvirus, molluscum contagiosum, papilloma viruses, parvovirus B19,rubella, measles and coxsackie A virus; and viruses that display atropism for genital tissue, include without limitation, herpes simplextype 2, papillomaviruses, human immunodeficiency virus (HIV).

In various embodiments, a method for treating a cancer in a subject inneed thereof is provided by administering an IRC described herein to thesubject wherein the peptide is a CD8+ epitope of a pathogen that has atropism for the tissue that is the source of the cancer (the “sourcetissue”). In various embodiments, the appropriate IRC is selected byfirst determining the source tissue of the tumor cell and then selectinga peptide: (1) to which the patient already has existing CD8+ T cells,and (2) that has a tropism for the source tissue of the tumor. Theselected IRC(s) are then administered to the subject in need thereof.

In various embodiments, provided are methods for treating a lung cancercomprising determining if a subject has been actively vaccinated againsta pathogen that infects lung cells, e.g., an influenza virus, e.g.,influenza virus type A or type B, then administering an effective amountof an IRC composition described herein, wherein the CD8+ T cell epitopeof the peptide is of the antigenic determinants of the pathogencontained in the vaccine and which T cell epitope forms a complex withan MHC molecule class I of the subject. The methods and uses describedherein for treating a lung cancer includes, in some embodiments,determining if a subject has been infected with pathogen that infectslung cells, e.g., an influenza virus, e.g., influenza virus type A ortype B, then administering an effective amount of an IRC compositiondescribed herein wherein the CD8+ T cell epitope of the peptide is ofthat pathogen and which T cell epitope forms a complex with an MHC classI molecule of the subject.

Provided also are methods for treating an oral cancer, which are part ofthe group of cancers commonly referred to as head and neck cancers, byadministering an IRC compositions described herein, wherein the CD8+epitope of the peptide is of a pathogen that has a tropism for oraltissue, e.g., a mumps virus, Epstein Barr virus, cytomegalovirus, or aherpes simplex virus type 1. The method comprises determining if asubject in need thereof has been actively vaccinated against, orinfected with, e.g., a mumps virus, Epstein Barr virus, cytomegalovirus,or a herpes simplex virus type 1, and if the subject has been vaccinatedor infected previously then administering to the subject an IRCcomposition described herein wherein the CD8+ epitope of the peptide isof a mumps virus or a measles virus or of the antigenic component of thevaccine the subject had received, or of the pathogen, i.e., mumps,measles, Epstein Barr virus, cytomegalovirus, or a herpes simplex virustype 1, that had previously infected the subject.

Combination Therapy

In various embodiments, the IRC compositions described herein areco-administered with other cancer therapeutics. Furthermore, in someembodiments, the IRCs described herein are administered in conjunctionwith other cancer treatment therapies, e.g., radiotherapy, chemotherapy,surgery, and/or immunotherapy. In some aspects of methods and usesdescribed herein, the IRC compositions described herein are administeredin conjunction with checkpoint inhibitors. In various embodiments thecapsid backbone is administered in conjunction with an immune agonist.In various embodiments, the IRC is administered in conjunction withtreatment with a therapeutic vaccine. In various embodiments, the IRC isadministered in conjunction with treatment with a conjugated antigenreceptor expressing T cell (CAR-T cell). In various embodiments, the IRCis administered in conjunction with treatment with anotherimmuno-oncology product. The IRCs of the present disclosure and othertherapies or therapeutic agents are, in some embodiments, administeredsimultaneously or sequentially by the same or different routes ofadministration. The determination of the identity and amount oftherapeutic agent(s) for use in the methods of the present disclosure isreadily made by ordinarily skilled medical practitioners using standardtechniques known in the art.

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties for allpurposes.

While the methods, uses, and compositions described herein have beenillustrated and described in detail in above, such illustration anddescription are to be considered illustrative or exemplary and notrestrictive. It will be understood that changes and modifications may bemade by those of ordinary skill within the scope and spirit of thefollowing claims. In particular, the present disclosure covers furtherembodiments with any combination of features from different embodimentsdescribed above and below.

The present disclosure is additionally described by way of the followingillustrative non-limiting examples that provide a better understandingof the present disclosure and of its many advantages. The followingexamples are included to demonstrate preferred embodiments of theinvention. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples which follow representtechniques used in the present disclosure to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the present disclosure.

EXAMPLES Example 1 Production of Truncated Mouse Papillomavirus (MPV1)L1 Protein

The truncated mouse papillomavirus L1 DNA sequence of 1138 base pairswas codon-optimized for E. coli expression and synthesized (SEQ IDNOS:135 and 136, two varieties of codon optimization) (GeneScriptBiotech, Piscataway, N.J.) and subsequently cloned into the T7expression vector Pet-24a(+) (MilliporeSigma, Burlington, Mass.). Thesequence was based on the wild type mouse (Mus musculus) papillomavirusL1 protein sequence except that it contains three deletion mutations atthree specific regions: one deletion at the amino-terminus (10 aminoacids removed), one at deletion the carboxy-terminus (34 amino acidsdeleted), and a third deletion in the helix four (H4) region close tothe carboxy-terminal region (deletion of amino acids 411 to 436 of theMPV L1 sequence). This mutant MPV L1 protein is hereinafter referred toas “MPV.10.34.d.” (See, FIG. 1B).

The wild type mouse (Mus musculus) L1 wild type protein sequence isdepicted in FIG. 1A and has the following protein sequence (SEQ ID NO:132, NCBI Reference Sequence: YP_003778198.1, DNA: 9434943):

Met Ala Met Trp Thr Pro Gln Thr Gly Lys Leu Tyr Leu Pro ProThr Thr Pro Val Ala Lys Val Gln Ser Thr Asp Glu Tyr Val TyrPro Thr Ser Leu Phe Cys His Ala His Thr Asp Arg Leu Leu ThrVal Gly His Pro Phe Phe Ser Val Ile Asp Asn Asp Lys Val ThrVal Pro Lys Val Ser Gly Asn Gln Tyr Arg Val Phe Arg Leu LysPhe Pro Asp Pro Asn Lys Phe Ala Leu Pro Gln Lys Asp Phe TyrAsp Pro Glu Lys Glu Arg Leu Val Trp Arg Leu Arg Gly Leu GluIle Gly Arg Gly Gly Pro Leu Gly Ile Gly Thr Thr Gly His ProLeu Phe Asn Lys Leu Gly Asp Thr Glu Asn Pro Asn Lys Tyr GlnGln Gly Ser Lys Asp Asn Arg Gln Asn Thr Ser Met Asp Pro LysGln Thr Gln Leu Phe Ile Val Gly Cys Glu Pro Pro Thr Gly GluHis Trp Asp Val Ala Lys Pro Cys Gly Ala Leu Glu Lys Gly AspCys Pro Pro Ile Gln Leu Val Asn Ser Val Ile Glu Asp Gly AspMet Cys Asp Ile Gly Phe Gly Asn Met Asn Phe Lys Glu Leu GlnGln Asp Arg Ser Gly Val Pro Leu Asp Ile Val Ser Thr Arg CysLys Trp Pro Asp Phe Leu Lys Met Thr Asn Glu Ala Tyr Gly AspLys Met Phe Phe Phe Gly Arg Arg Glu Gln Val Tyr Ala Arg HisPhe Phe Thr Arg Asn Gly Ser Val Gly Glu Pro Ile Pro Asn SerVal Ser Pro Ser Asp Phe Tyr Tyr Ala Pro Asp Ser Thr Gln AspGln Lys Thr Leu Ala Pro Ser Val Tyr Phe Gly Thr Pro Ser GlySer Leu Val Ser Ser Asp Gly Gln Leu Phe Asn Arg Pro Phe TrpLeu Gln Arg Ala Gln Gly Asn Asn Asn Gly Val Cys Trp His AsnGlu Leu Phe Val Thr Val Val Asp Asn ThrArg Asn Thr Asn Phe Thr Ile Ser Gln Gln Thr Asn Thr Pro AsnPro Asp Thr Tyr Asp Ser Thr Asn Phe Lys Asn Tyr Leu Arg HisVal Glu Gln Phe Glu Leu Ser Leu Ile Ala Gln Leu Cys Lys ValPro Leu Asp Pro Gly Val Leu Ala His Ile Asn Thr Met Asn ProThr Ile Leu Glu Asn Trp Asn Leu Gly Phe Val Pro Pro Pro GlnGln Ser Ile Ser Asp Asp Tyr Arg Tyr Ile Thr Ser Ser Ala ThrArg Cys Pro Asp Gln Asn Pro Pro Lys

Likewise, the wild type nucleic acid sequence for MPV1 L1 protein (SEQID NO: 133) is as follows:

ATGGCAATGTGGACACCCCAGACCGGGAAGCTTTACCTCCCACCTACAACTCCAGTGGCAAAAGTGCAGAGCACAGACGAATATGTGTACCCTACGTCTCTCTTCTGTCATGCACACACGGACCGTTTGCTAACAGTGGGCCACCCTTTTTTTTCTGTCATTGACAATGACAAGGTCACTGTGCCTAAAGTGTCTGGCAACCAATATAGGGTTTTCAGACTTAAATTCCCAGATCCAAATAAATTTGCATTGCCCCAAAAGGATTTCTATGATCCTGAGAAAGAACGGTTAGTGTGGAGGTTAAGGGGTCTGGAAATTGGAAGAGGTGGCCCATTAGGGATTGGCACTACCGGGCACCCCCTTTTTAACAAGCTTGGAGACACGGAAAATCCAAATAAATATCAGCAAGGCTCTAAGGATAATAGGCAGAACACTTCCATGGACCCCAAACAAACACAGCTGTTTATTGTTGGCTGTGAACCCCCTACAGGGGAACACTGGGATGTAGCTAAGCCCTGTGGAGCTCTGGAGAAGGGTGACTGCCCTCCTATCCAACTTGTAAATAGTGTAATTGAGGATGGGGATATGTGTGACATTGGCTTTGGGAATATGAACTTCAAAGAGCTGCAGCAGGATAGGAGTGGTGTGCCTCTTGATATTGTATCTACCCGGTGCAAATGGCCCGACTTTCTGAAAATGACCAATGAGGCATATGGGGATAAGATGTTCTTCTTTGGAAGGAGAGAGCAAGTGTATGCAAGACACTTTTTCACCAGGAATGGCTCTGTGGGGGAGCCCATACCAAACTCTGTGAGTCCCAGTGACTTTTACTACGCACCCGACAGCACACAGGACCAGAAGACACTCGCACCCTCCGTGTACTTTGGAACTCCTAGTGGGTCACTTGTGTCGAGTGATGGTCAGCTGTTTAACAGGCCATTTTGGCTTCAAAGGGCTCAGGGAAACAATAATGGTGTGTGCTGGCACAATGAGCTCTTTGTTACTGTTGTCGACAACACAAGGAATACAAACTTTACTATCTCCCAGCAAACCAACACACCAAACCCAGATACATATGACTCTACTAATTTTAAAAACTATTTAAGACATGTGGAACAATTTGAGCTGTCCCTTATTGCTCAACTGTGTAAGGTTCCACTTGACCCGGGTGTGCTTGCCCATATAAACACTATGAACCCAACCATCTTGGAGAACTGGAACTTGGGTTTTGTACCTCCCCCACAGCAGTCCATCTCTGATGACTATAGGTATATAACATCATCGGCAACTCGCTGTCCAGATCAGAATCCGCCCAAGGAAAGAGAGGATCCTTACAAGGGTCTTATATTTTGGGAAGTTGATCTTACTGAGAGGTTTTCTCAGGACCTTGATCAGTTTGCTCTGGGACGAAAGTTTCTGTATCAAGCTGGTATACGTACTGCTGTTACGGGCCGCGGGGTCAAAAGGGCAGCGTCTACAACCTCTGCGTCTTCTAGACGAGTTGTAAAACGGAAGAGGGGAAGCAAATAA

In contrast, the mutant MPV sequence selected for the following studiesis depicted in FIG. 1B and has the following amino acid sequence (SEQ IDNO:134):

Met Leu Tyr Leu Pro Pro Thr Thr Pro Val Ala Lys Val Gln SerThr Asp Glu Tyr Val Tyr Pro Thr Ser Leu Phe Cys His Ala HisThr Asp Arg Leu Leu Thr Val Gly His Pro Phe Phe Ser Val IleAsp Asn Asp Lys Val Thr Val Pro Lys Val Ser Gly Asn Gln TyrArg Val Phe Arg Leu Lys Phe Pro Asp Pro Asn Lys Phe Ala LeuPro Gln Lys Asp Phe Tyr Asp Pro Glu Lys Glu Arg Leu Val Trp Arg Leu Arg Gly LeuGlu Ile Gly Arg Gly Gly Pro Leu Gly Ile Gly Thr Thr Gly HisPro Leu Phe Asn Lys Leu Gly Asp Thr Glu Asn Pro Asn Lys TyrGln Gln Gly Ser Lys Asp Asn Arg Gln Asn Thr Ser Met Asp ProLys Gln Thr Gln Leu Phe Ile Val Gly Cys Glu Pro Pro Thr GlyGlu His Trp Asp Val Ala Lys Pro Cys Gly Ala Leu Glu Lys GlyAsp Cys Pro Pro Ile Gln Leu Val Asn Ser Val Ile Glu Asp GlyAsp Met Cys Asp Ile Gly Phe Gly Asn Met Asn Phe Lys Glu LeuGln Gln Asp Arg Ser Gly Val Pro Leu Asp Ile Val Ser Thr ArgCys Lys Trp Pro Asp Phe Leu Lys Met Thr Asn Glu Ala Tyr GlyAsp Lys Met Phe Phe Phe Gly Arg Arg Glu Gln Val Tyr Ala ArgHis Phe Phe Thr Arg Asn Gly Ser Val Gly Glu Pro Ile Pro AsnSer Val Ser Pro Ser Asp Phe Tyr Tyr Ala Pro Asp Ser Thr GlnAsp Gln Lys Thr Leu Ala Pro Ser Val Tyr Phe Gly Thr Pro SerGly Ser Leu Val Ser Ser Asp Gly Gln Leu Phe Asn Arg Pro PheTrp Leu Gln Arg Ala Gln Gly Asn Asn Asn Gly Val Cys Trp HisAsn Glu Leu Phe Val Thr Val Val Asp Asn Thr Arg Asn Thr AsnPhe Thr Ile Ser Gln Gln Thr Asn Thr Pro Asn Pro Asp Thr TyrAsp Ser Thr Asn Phe Lys Asn Tyr Leu Arg His Val Glu Gln PheGlu Leu Ser Leu Ile Ala Gln Leu Cys Lys Val Pro Leu Asp ProGly Val Leu Ala His Ile Asn Thr Met Asn Pro Thr Ile Leu GluAsn Trp Asn Leu Gly Phe Val Pro Pro Lys Glu Arg Glu Asp ProTyr Lys Gly Leu Ile Phe Trp Glu Val Asp Leu Thr Glu Arg PheSer Gln Asp Leu Asp Gln Phe Ala Leu Gly Arg Lys Phe Leu Tyr Gln

Alignment of the wild type sequence with the triple truncationMPV.10.34.d sequence is shown in FIG. 2. Additionally, the nucleic acidsequence (below) of MPV.10.34.d was optimized for expression. Thesequence was optimized for codon usage within the target host as well asfor expression level to maximize expression efficiency within the host.Below are provided two alternative optimized nucleic acid sequences forMPV.10.34.d used herein (SEQ ID NO: 135):

ATGCTGTACCTGCCGCCGACCACCCCGGTGGCGAAAGTTCAGAGCACCGACGAATACGTTTATCCGACCAGCCTGTTCTGCCACGCGCACACCGATCGTCTGCTGACCGTGGGTCACCCGTTCTTTAGCGTTATCGACAACGATAAGGTGACCGTTCCGAAAGTGAGCGGCAACCAGTACCGTGTTTTTCGTCTGAAGTTCCCGGACCCGAACAAATTTGCGCTGCCGCAAAAGGACTTCTATGATCCGGAGAAGGAACGTCTGGTGTGGCGTCTGCGTGGTCTGGAAATTGGTCGTGGTGGCCCGCTGGGTATTGGTACCACCGGTCACCCGCTGTTCAACAAACTGGGCGATACCGAGAACCCGAACAAATATCAGCAAGGTAGCAAGGACAACCGTCAGAACACCAGCATGGACCCGAAGCAGACCCAACTGTTTATTGTTGGTTGCGAGCCGCCGACCGGTGAACACTGGGATGTTGCGAAACCGTGCGGTGCGCTGGAAAAGGGCGATTGCCCGCCGATCCAACTGGTGAACAGCGTTATTGAGGACGGTGATATGTGCGACATCGGTTTTGGCAACATGAACTTCAAAGAACTGCAGCAAGACCGTAGCGGCGTGCCGCTGGATATTGTTAGCACCCGTTGCAAATGGCCGGACTTCCTGAAGATGACCAACGAAGCGTACGGTGATAAGATGTTCTTTTTCGGCCGTCGTGAGCAGGTTTATGCGCGTCACTTTTTCACCCGTAACGGTAGCGTGGGCGAGCCGATCCCGAACAGCGTTAGCCCGAGCGACTTCTACTATGCGCCGGACAGCACCCAGGATCAAAAAACCCTGGCGCCGAGCGTGTACTTTGGTACCCCGAGCGGCAGCCTGGTTAGCAGCGATGGTCAACTGTTTAACCGTCCGTTCTGGCTGCAGCGTGCGCAGGGTAACAACAACGGCGTGTGCTGGCACAACGAACTGTTTGTTACCGTGGTTGACAACACCCGTAACACCAACTTCACCATCAGCCAGCAAACCAACACCCCGAACCCGGACACCTACGATAGCACCAACTTTAAAAACTATCTGCGTCACGTGGAGCAGTTCGAACTGAGCCTGATTGCGCAACTGTGCAAAGTGCCGCTGGACCCGGGTGTGCTGGCGCACATCAACACCATGAACCCGACCATTCTGGAGAACTGGAACCTGGGTTTCGTTCCGCCGAAAGAGCGTGAAGACCCGTACAAGGGCCTGATCTTCTGGGAAGTGGATCTGACCGAACGTTTCAGCCAGGACCTGGATCAATTTGCGCTGGGCCGTAAATTCCTGTATCAGTAA And (SEQ ID NO: 136):GAATTGGCGGAAGGCCGTCAAGGCCACGTGTCTTGTCCGCGGTACCCATATGCTGTATCTGCCTCCAACTACACCGGTTGCAAAAGTTCAGAGCACCGATGAATATGTTTATCCGACCAGCCTGTTTTGTCATGCACATACCGATCGTCTGCTGACCGTTGGTCATCCGTTTTTTAGCGTTATTGATAACGATAAAGTGACCGTTCCGAAAGTTAGCGGTAATCAGTATCGTGTTTTTCGCCTGAAATTTCCGGATCCGAACAAATTTGCACTGCCGCAGAAAGATTTTTACGACCCGGAAAAAGAACGTCTGGTTTGGCGTCTGCGTGGTCTGGAAATTGGTCGTGGTGGTCCGTTAGGTATTGGCACCACCGGTCATCCGCTGTTTAACAAACTGGGTGATACCGAAAATCCGAATAAATACCAGCAGGGCAGCAAAGATAATCGTCAGAATACCAGTATGGATCCGAAACAGACCCAGCTGTTTATTGTTGGTTGTGAACCGCCTACCGGTGAACATTGGGATGTTGCAAAACCGTGTGGTGCACTGGAAAAAGGTGATTGTCCGCCTATTCAGCTGGTTAATAGCGTGATTGAAGATGGTGATATGTGCGATATTGGCTTTGGCAACATGAACTTTAAAGAACTGCAGCAGGATCGTAGCGGTGTTCCGCTGGATATTGTTAGCACCCGTTGTAAATGGCCTGATTTTCTGAAAATGACCAATGAAGCCTATGGCGACAAAATGTTTTTTTTCGGTCGTCGTGAACAGGTTTATGCCCGTCACTTTTTTACCCGTAATGGTAGCGTTGGTGAACCGATTCCGAATAGCGTTAGCCCGAGCGATTTCTATTATGCACCGGATAGCACCCAGGATCAGAAAACCCTGGCACCGAGCGTTTATTTTGGCACCCCGAGCGGTAGCCTGGTTAGCAGTGATGGTCAGCTGTTCAATCGTCCGTTTTGGCTGCAGCGTGCACAGGGTAATAACAATGGTGTTTGTTGGCATAACGAACTGTTTGTTACCGTTGTTGATAATACCCGCAATACCAACTTTACCATTAGCCAGCAGACCAATACACCGAATCCGGATACCTATGATAGCACCAACTTCAAAAACTATCTGCGTCATGTGGAACAGTTTGAACTGAGCCTGATTGCCCAGCTGTGTAAAGTGCCGCTGGATCCGGGTGTTCTGGCACATATTAACACCATGAATCCGACCATTCTGGAAAATTGGAATCTGGGTTTTGTTCCGCCTAAAGAACGTGAAGATCCGTATAAAGGTCTGATTTTTTGGGAAGTTGATCTGACCGAACGTTTTAGCCAGGATCTGGATCAGTTTGCACTGGGTCGCAAATTTCTGTATCAGTAACTCGAGGAGCTCGGAGCACAAGACTGGCCTCATGGGCCTTCCGCTCACTGC C

The general protocol for recombinant expression and purification of themutant MPV.10.34.d is schematically depicted in FIG. 3.

The MPV.10.34.d nucleic acid sequence was generated from wild type mousepapillomavirus sequence via site-mutagenesis (Genscript Biotech,Piscataway, N.J.) using the following primer sequence (SEQ ID NO: 137):

AAGCTTGTCGACGGAGCTCGAATTCGGATCCTTATTACTGATACAGGAATT TACGGCCCAGC

The MPV.10.34.d nucleic acid sequence was then cloned into themulticloning site of expression vector pet24a(+) (MilliporeSigma,Burlington, Mass.) using restriction endonucleases Ndel and BamH1according to standard protocols. The correct cloning into the multiplecloning site and construct sequence was confirmed by both restrictionendonuclease enzyme digestion using MIu1 and BamH1 as well as Sangersequencing using both T7 forward and reverse primers.

Expression was achieved by transforming the pet24a(+) plasmid containingMPV.10.34.d into T7 expression competent Escherichia coli 2566 cells(New England Biolabs, Ipswich, Mass., US), and colony selection on solidmedia. A single colony was grown according to standard protocols inLurea broth (LB) media. Briefly, 5 mL sterile LB including 50 μg/mLkanamycin (Quality Biological, Gaithersburg, Md., US) was seeded with asingle colony selected from the solid media and grown overnight at 37°C. with shaking. The seed culture was then diluted 1:25 and growth wascontinued at 37° C. until OD600 reached about 0.6 to 0.8. Then about 1mM final concentration of isopropyl β-d-1-thiogalactopyranoside (IPTG,Invitrogen, Carlsbad, Calif., US) was added to the culture to induceexpression from the plasmid. Induction was continued under theseconditions for an additional four hours after which cell pellets arecollected by centrifugation at 4000×g for 15 minutes at 4° C. Thesupernatant was discarded and the cell pellets were stored at −20° C.unless immediately used.

MPV.10.34.d was expressed as inclusion bodies (IBs). To recover IBMPV.10.34.d, pellets were first thawed (if frozen) and then resuspendedin 20 mL per 1 L pellet lysis buffer (50 mM Tris, pH 8.0, 500 mM NaCl, 1mM EDTA, 1 mM protease inhibitor phenylmethylsulfonyl fluoride (PMSF).Resuspended material was then homogenized using a high pressuredhomogenizer (Avestin Emulsiflex C3™, ATA Scientific, Taren Point,Australia) and cells were passed through the homogenizer and lysed 4times at about 15,000 to 20,000 PSI. The lysed bacterial cells were thencentrifuged at 25,000×g at 4° C. for 20 min. Supernatant was thendiscarded and the inclusion body pellet was stored at −20° C.

Next the IB were solubilized by resuspending the pellet (50 mL per 1 Lpellet) in of 6 M urea buffer (8 M Urea, 50 mM Tris, pH 8.0, 500 mMNaCl, 1 mM EDTA, 1 mM PMSF, and 1 mM DTT). Resuspended contents wereonce more passaged three to four times through the homogenizer (AvestinEmulsiflex C3™, ATA Scientific, Taren Point, Australia) at about 15,000to 20,000 PSI. The resolubilized samples were centrifuged at 25,000×g at4° C. for 20 min. The supernatant was collected into a container that issufficiently large enough to hold the volume of a sample. The pellet wasdiscarded. The supernatant was stored at 4° C. or −20° C.

Following solubilization, the MPV.10.34.d was refolded by removal of thedenaturant (6M Urea) in a step-gradient manner. The solubilized sampleswere inserted into dialysis tubing (snakeskin dialysis tubing, 10,000 Damolecular weight cut off, 35 mm. (ThermoFisher Scientific, Waltham,Mass., US). In general, about 100 to about 150 mL of resolubilizedsample solution was dispensed into a single dialysis tube. The sampleswere first dialyzed (sample to buffer ratio 1:12.5) against 4 M ureabuffer (50 mM Tris, pH 8.0, 500 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM DTT,and 0.05% Tween®-80) for 3±1 hour in a cold room at about 4° C. on astir plate. Then, the samples were again dialyzed against a fresh 1 Murea buffer (50 mM Tris, pH 8.0, 500 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mMDTT, and 0.05% Tween-80) for 3±1 hour in a cold room on a stir plate.Subsequently, the samples were dialyzed against 0 M urea buffer (50 mMTris, pH 8.0, 500 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM DTT, and 0.05%Tween-80) overnight (about 16 to 18 hours) in a cold room at about 4° C.on a stir plate. The dialyzed/refolded sample solutions were aliquotedinto 50 mL conical tubes and stored in a −20° C. freezer.

To obtain a MPV.10.34.d of greater than 95% purity for subsequentmedicinal use, samples were subjected to a two-step chromatographypurification which involves a capture step utilizing cation exchangechromatography (CEX) followed by a polishing step using a hydrophobicinteraction column (HIC). For the capture step, the refolded MPV.10.34.dsamples were removed from the −20 and thawed on ice. Next, the samplewas dialyzed into capture buffer A (25 mM NaPO₄, 25 mM NaCl, pH 6.0).Following dialysis, samples were centrifuged 4000×g, for about 10 min,at 4° C. and then filtered through a 0.22 μm polyethersulfone (PES)membrane. The refolded MPV.10.34.d protein was then captured by CEX(Fractogel® EMD S03-M, EMD Millipore, Burlington, Mass., US) and thenstep eluted with 30% 25 mM NaPO₄, 1.5 M NaCl, pH 6.0. This resulted inpurified refolded MPV.10.34.d of purity of at least 80%.

To further remove contaminants and increase purity of the MPV.10.34.d toabove 95%, the CEX eluate was diluted with high-salt buffer to achieveloading conditions of 25 mM NaPO₄, 3 M NaCl, pH 6.0, and applied to HICresin (butyl-S-Sepharose® Fast Flow, GE Healthcare Life Sciences/FisherScientific, Waltham, Mass., US). The bound refolded MPV.10.34.d productwas subjected to a pre-elution wash with 30% 25 mM NaPO₄, 25 mM NaCl, pH6.0, and then eluted with a single step gradient of 70% 25 mM NaPO₄, 25mM NaCl, pH 6.0. Greater than 95% purity MPV.10.34.d was stored in a−20° C. freezer in the elution buffer.

Greater than 95% purity MPV.10.34.d was confirmed via SDS-PAGE followedby Coomassie blue gel and silver staining. For Coomassie staining, gelswere incubated in water to remove SDS-PAGE running buffer, thenincubated for 5 minutes in SimplyBlue SafeStain (Novex, Carlsbad,Calif.). Gels were de-stained in water. (See photographs of gels in FIG.4A). Silver staining was performed using a Pierce Silver stain kit(ThermoFisher Scientific, Rockford, Ill.) according to manufacturer'sinstructions. (See, FIG. 4B). To estimate purity, the images of the gelswere taken using the Bio-Rad Image Lab 6.01 software. Gel images werethen uploaded into the software and the entire vertical lane containingthe band of interest (“lane profile”) was analyzed using the imageanalysis software. The specific total density of the band of the proteinof interest was calculated by drawing a box or freehand shape around theband. Subsequently, the total density of the entire lane was measured inthe same manner. After obtaining the measurements, the backgrounddensity of a suitably matched area on the gel in each case wassubtracted. This background-corrected density of the protein band by thebackground-corrected density of the whole lane was then multiplied by100 to obtain the percent purity.

From this analysis and as seen in both FIGS. 4A and 4B, the main processsteps described above provided incremental purification of the ˜50 kDAMPV.10.34.d protein. Non-specific proteins above and below the 50 kDAband were significantly reduced across each of the purification steps,where lane 1 was a post-cell harvest sample, wash, and homogenization;lane 2 was a post-IB solubilization sample, lane 3 was a post-refoldingsample, lane 4 was a post-capture chromatography via CEX sample, andlane 5 was a post-polishing step sample using HIC.

Example 2 Determination of MPV.10.34.d Structure and Size

DLS (dynamic light scattering) and TEM revealed that upon refoldingMPV.10.34.d unexpectedly forms capsid backbones that are about 20 nm to30 nm in diameter. (See, FIG. 5). To analyze the refolded MPV.10.34.dsamples, the purified samples were first analyzed by DLS to obtaindetermine whether refolding of MPV.10.34.d occurred.

60 μl of sample was placed in a 40 μL solvent-resistant micro-cuvette(ZEN0040, Malvern Panalytical, Waltham, Mass.) and the cell wassubsequently placed into a Zetasizer Nano ZS Dynamic Light scatteringinstrument (Malvern Panalytical, Waltham, Mass.). This was aresearch-grade dynamic light scattering system for measurement ofprotein size, electrophoretic mobility of proteins, zeta potential ofcolloids and nanoparticles, and optionally the measurement of proteinmobility, and microrheology of protein and polymer solutions. The highperformance of the Zetasizer Nano ZS also enables the measurement of themolecular weight and second virial coefficient, A2, of macromoleculesand kD, the DLS interaction parameter. The system can also be used in aflow configuration to operate as a size detector for SEC or FFF. Once inthe machine, the sample was processed with the companion software(Zetasizer Nano Software, Malvern Panalytical). The program was set toread the sample for a total of 5 runs to generate two plots.

The two plots generated were determine capsid backbone size andstructure, intensity (FIG. 6A), and volume (FIG. 6B). The intensitydistribution provides the amount of light scattered by the particles inthe different sized bins. The volume distribution demonstrates the totalvolume of particles in various sized bins. In other words, the intensityplot (FIG. 6A) provides an assessment of the overall population sizes ofMPV.10.34.d particles including host cell contaminants within thesample, whereas the volume plot (FIG. 6B) determines the relativeproportion of refolded protein with respect to other contaminants, i.e.,host bacterial cell proteins.

Note that each individual plot line in the graphs of FIGS. 6A and 6Brepresent individual samples (five samples for each of A and B). If thevolume curve was between about 10 to 15 nm (X-axis), the shell wasdetermined to be a capsomer made up of 5 MPV.10.34.d units. For T=1icosahedral capsid backbones made up of 60 MPV.10.34.d, the volume curveplot is about 20 to 30 nm. For T=7 icosahedral capsid backbones made upof 360 MPV.10.34.d, the volume curve plot was about 50 to 60 nm. As isreadily apparent from the intensity graph (FIG. 6A), there are twopeaks, one lower at approximately 20 to 30 nm and one atapproximately >100 nm, whereas the volume graph (FIG. 6B) shows a singlesize of about 20 nm to 30 nm. It was postulated that the two peaks onthe intensity figure were attributable to the presence of distinctpopulations of capsid backbones that arose after refolding of theMPV.10.34.d into the capsid backbone since the samples at this stage hadnot undergone purification. However, the larger of the two peaks shownin the intensity plot constitute only a small portion of the sample, anda majority of the sample falls within the smaller peak. Hence, the DLSresults show that a majority of the MPV.10.34.d are structures that areabout 20 nm to 30 nm in size.

In DLS, information regarding the motion (diffusion) of submicronparticles in a solution is extracted from the rate of scatteringintensity fluctuations using a statistical technique called intensityautocorrelation. The mean particle size and distribution are calculatedfrom the distribution of diffusion coefficients using the StokesEinstein equation. Because the magnitude of the scattering intensityvaries roughly with the 6^(th) power of the particle size, DLS is highlysensitive to the presence of small amounts of aggregates in a mixture ofcapsid backbones which is believed to be reflected in the intensity plotshown in FIG. 6A.

TEM analysis was also employed to obtain further visual confirmation ofthe structure and size of the refolded proteins. Samples (10 μL) wereadsorbed to glow discharged (EMS GloQube) carbon coated 400 mesh coppergrids (EMS), by floatation for 2 min. Grids were quickly blotted andthen rinsed in 3 drops (1 min each) of TBS. Grids were negativelystained in 2 consecutive drops of 1% uranyl acetate with tylose (1% UAT,double filtered, 0.22 μm filter), blotted then quickly aspirated toobtain a thin layer of stain covering the sample. Grids were imaged on aPhillips CM-120 TEM operating at 80 kV with an AMT XR80 CCD (8megapixel).

TEM results revealed that MPV.10.34.d formed capsid backbones that had amarkedly grooved appearance, with pentagonal/capsomer “towers.” (See,FIG. 5). Measurements showed that these capsid backbones wereapproximate 30 nm is size (compare with the 500 nm scale on TEMmicrograph, FIG. 5).

These results were unexpected because deletion of residues in the helixfour H4 region of L1 has been reported to not lead to T=1 geometrycapsid backbone formation. Further, it has been shown that the samedeletions result in capsomers of T=7 in HPV11 and HPV16 L1 proteins.(See, Chen et al., Mol. Cell, 5:557-567, 2000, WO 2000054730, Bishop etal., Virol. J., 4:3, 2007, and Schädlich et al., J. Virol.,83(15):7690-7705, 2009). In summary, these findings support theconclusion that the MPV.10.34.d constructs form icosahedral capsidbackbones of T=1 lattice geometry comprised of 60 monomers, or 12capsomers.

Example 3 MPV.10.34.d Capsid Backbone Assembly Requires Reductant

HPV particles form T=7 geometry particles that are 50 to 60 nm isdiameter. The manufacture and production of such capsid backbones ineither eukaryotic or prokaryotic host cell systems involves theexpression in suitable host cell system followed by a series ofpurification step to yield highly purified capsid backbones. Thesecapsid backbones are then subjected to a disassembly and re-assembly(DARA) step. Briefly, this involves the addition of DTT or a similarreducing agent to disassemble the capsid backbones intocapsomers/pentamers (made from five L1 monomers) and then removal of thereducing agent for assembly back to T=7 capsid backbones that have beendocumented as more symmetric and stable.

To further delineate how MPV.10.34.d refolds into a T=1 capsid backbone,the refolding steps in Example 1 were repeated. Briefly, followingsolubilization of the IB, MPV.10.34.d was subjected to refolding viaremoval of the denaturant (6M Urea) in a step-gradient manner. Thesolubilized samples were inserted into dialysis tubing (snakeskindialysis tubing, 10,000 Da molecular weight cut off 35 mm. (ThermoFisherScientific, Waltham, Mass., US). In general, about 100 mL to about 150mL of resolubilized sample solution was dispensed into a four dialysistubes. The samples were then dialyzed against 4 M urea buffer with thegeneral buffer recipe 50 mM Tris, pH 8.0, 500 mM NaCl and 0.05% Tween-80for 3±1 hour in a cold room at about 4° C. on a stir plate. Then thesamples were again dialyzed against a fresh 1 M urea buffer for 3±1 hourin a cold room on a stir plate. Subsequently, the samples were dialyzedagainst 0 M urea buffer overnight (about 16 to 18 hours) in a cold roomat about 4° C. on a stir plate.

The main difference between the four buffer conditions in thisexperiment were the presence of: (i) 1 mM EDTA, 1 mM PMSF, and 1 mM DTT;(ii) 1 mM EDTA, (iii) 1 mM DTT, and (iv) no added ETDA, PMSF or DTT. Thedialyzed/refolded sample solutions under all four conditions werealiquoted into 50 mL conical tubes and analyzed via DLS as described inExample 2 before being stored in a −20° C. freezer. As shown in FIG. 7using volume plots (and as explained in Example 2), aggregates as markedby the arrow were observed in sample (ii) and sample (iv). (See, FIG. 7Cand FIG. 7D, respectively). Little to no aggregates were observed on theDLS volume plots in sample (i) or sample (iii). (See, FIG. 7A and FIG.7B, respectively).

To further confirm whether MPV.10.34.d successfully refolded into T=1capsid backbones, all samples were dialyzed into capture buffer A (25 mMNaPO₄, 25 mM NaCl, pH 6.0) overnight at 4° C. Following dialysis,samples were centrifuged at 4000×g for 10 min at 4° C. and then filteredthrough a 0.22 μm PES membrane. The T=1 capsid backbone platform wasthen captured by CEX (EMD Fractogel S03 (M), EMD Millipore, Darmstadt,Germany) and then step eluted with 30% 25 mM NaPO₄, 1.5M NaCl, pH 6.0.Results are shown in FIG. 8. FIG. 8 shows successful capture and elutionof correctly refolded T=1 capsid backbones in samples that were eitherrefolded in DTT/ETDA/PMSF (FIG. 8A) or DTT alone (FIG. 8B). In contrast,there was little to no capture and elution of correctly refolded T=1capsid backbones under refolding conditions that included ETDA alone(FIG. 8C) or no additives (FIG. 8D) as demarcated by the arrows in FIG.8.

Taken together, the data indicate that the process of producing T=1capsid backbones favors reducing conditions. This contrasts with theknown process of producing HPV T=7 capsid backbones that require theeventual removal of any reducing agents for successful capsid backboneassembly.

Example 4 Production of Soluble MPV.10.34.d

Bacterial expression of MPV.10.34.d at 37° C. for 4 hours in thepresence of 1 mM IPTG results in the expression and formation of IBsthat must be treated in a series of process steps as described inExample 1 to obtain highly purified 20 nm to 30 nm T=1 capsid backbonestructures. To investigate whether soluble MPV.10.34d can be expressedintracellularly in E. coli and whether it can assemble into a T=1 capsidbackbone as opposed to an IB, a strategy was adopted to lower theinduction temperature and IPTG concentrations to slow down expression,and thereby to prevent formation of IBs.

From Example 1, induction temperature was lowered from 37° C. to 16° C.at a concentration of IPTG of 100 μM and 1 mM. Briefly, freshly pickedE. coli C2566 colonies transformed with the MPV.10.34d DNA wereinoculated into LB with 50 μg/mL of kanamycin (henceforth LB+KAN)starter cultures, grown overnight at 37° C. and 250 rpm. The next daythe starter culture was used to inoculate a fresh 250 mL LB+KAN cultureat 1:25 dilution and grown at 37° C., 250 rpm, for about 1.5 to 2 hours,such that the culture reached an OD600 of about 0.5 to 0.7. At thispoint, the culture was induced with 1 mM and other tested IPTGconcentration(s) and shaken at 16° C. induction temperature overnight.The next day, post-induction samples (1 mL each) were pelleted at 1500×gfor 10 min. The supernatant was removed and the remaining bulk sampleswere pelleted at 4000×g for 10 min at 4° C. These samples were stored at−20° C. until analysis.

To determine soluble material, cell were lysed using a homogenizer(15,000-20,000 psi, 3 cycles). Samples of uninduced, induced, post-lysissoluble, and post-lysis insoluble material were analyzed by SDS-PAGE andWestern blot. Results show that lowering the induction temperature to16° C. resulted in undetectable levels of protein (Data not shown).

Since expression of MPV.10.34.d was low to undetectable at 16° C.,induction was attempted at a higher temperature, 25° C. At an inductiontemperature of 25° C., MPV.10.34d was successfully expressed. A majorityof MPV.10.34.d was expressed in the form of IBs (data not shown). Athird attempt at 30° C. was attempted under the induction conditionsdescribed above. It was discovered that soluble expression improvedtremendously at this temperature. There appeared to be a criticaltransition between 25° C. and 30° C. that allows for MPV.10.34.d to besuccessfully translated with approximately 50% expressed in a solubleform. In contrast, the concentration of IPTG appeared to have a minoreffect in the amount of translated MPV.10.34.d.

To purify these soluble MPV.10.34.d, a single step elutionchromatography purification was developed and subsequently employed.Briefly, soluble lysate containing MPV.10.34d was prepared in 50 mMNaPO₄, 50 mM NaCl, pH 7.0. A 1 mL prepacked Fractogel SO3 (M) column wasequilibrated to 50 mM NaPO₄, 50 mM NaCl, pH 7.0 and 500 μL of MPV.10.34dlysate was injected onto the column. A series of step gradient elutionswere performed to identify a conductivity window for elution ofMPV.10.34d, including 5, 10, 15, 20, 25, and 100% B steps (B=50 mMNaPO₄, 1.5M NaCl, pH 7.0; which translates to a theoretical NaClconcentration of 0.075M, 0.15M, 0.225M, 0.3M, 0.375M, and 1.5M). Basedon the elution of MPV.10.34d, a single step method was developed andscaled up to a 5 mL prepacked Fractogel SO3 (M) column with injectionvolumes of up to 5 mL. This single step method used 15% B, where B was50 mM NaPO₄, 500 mM NaCl, pH 7.0. (The lower concentration of NaClallowed for more consistent control of the conductivity, as relying onsmall percentage changes of a 1.5M NaCl resulted in conductivityirregularities when using 1 mL columns, likely due to pre-column mixingvolumes).

Based on these efforts, a single step gradient elution method wasdeveloped to generate milligram-scale amounts of CEX-capturedMPV.10.34d. Elution material was subsequently collected and analyzed asdescribed in Example 2. Results revealed that DLS (FIGS. 9A and 9B) andTEM data (FIG. 9C) from eluted MPV.10.34d yielded T=1 capsid backbonesof 20 to 30 nm diameter.

Example 5 Soluble and IB MPV.10.34.D Form T=1 Capsid Backbones

The MPV.A4 antibody is a conformational antibody that specifically bindsto MPV L1 in the form of T=1 or T=7 capsid backbone structure. Thisantibody will not bind to denatured or monomeric MPV L1. (Hafenstein etal., 2020, “Atomic Resolution CRYOEM structure of Mouse Papillomavirus,”International Papillomavirus Conference, July 20-24, 2020). To determinewhether MPV.10.34.d undergoing the steps in Example 1 or Example 4yields a T=1 capsid backbone, ELISA was performed on these samples withthe MPV.A4 monoclonal antibody.

Samples from Example 1 and Example 4 of equal concentrations (startingconcentration of 1000 ng/well) were subjected to ELISA. To ensure thatboth soluble and refolded MPV.10.34.d were equally bound to the ELISAplate (Nunc Maxisorp, ThermoFisher Scientific, Waltham, Mass., US), bothsamples were first buffer exchanged into either 50 mM NaPO₄, 450 mM NaClat pH 6 or pH 7. This resulted in two different pH conditions for bothsamples. Based on this, a total of four sample conditions were two-foldserially diluted and subjected to ELISA with the MPV.A4 monoclonalantibody.

Briefly, eight different amounts of protein (7.8 ng to 1μ.g) for eachsample under both pH conditions (into either 50 mM NaPO₄, 450 mM NaCl atpH 6 or pH 7) were first added to the ELISA plate and the plate wasstored at 4° C. Two days later, ELISA was performed by incubating eachplate for one hour at room temperature on an orbital shaker (300 rpm)with MPV.A4 mAb diluted 1:1000 using blocking buffer (4% dry milk, 0.2%Tween-20) and the plates incubated for one hour at 4° C. A wash step wasthen employed using wash buffer (0.35 M NaCl, 1.5 mM KH₂PO₄, 6.5 mMNa₂HPO₄, 0.05% Tween-20) at room temperature for a total of three washes(200 μL per sample per wash). Following the wash step, a goat anti-mouseIgG-HRP antibody (Millipore Sigma, St. Louis, Mo., US) was added at1:7000 dilution in blocking buffer (4% dry milk, 0.2% Tween-20) to afinal concentration of 82.9 ng/mL and the plates incubated for one hourat room temperature on an orbital shaker (300 rpm). After theincubation, the plate was washed and incubated with a peroxidasesubstrate (3, 3′, 5, 5′ tetramethyl benzidine, SeraCare Life Sciences,Inc., Milford, Mass., US) for 30 minutes, followed by the addition andincubation of stop solution (0.36 N H2504) (J.T. Baker/Avantor,Allentown, Pa., US) for 20 minutes. The absorbance of the sample plateswere read at 450 nm and 620 nm with a plate reader (BioTek, Winooski,Vt., US).

Results (FIG. 10) showed that the undialyzed soluble MPV10.34.d capsidbackbone, which are captured using buffer at pH 7 (solid circles), aswell as the soluble form dialyzed against buffer at pH 7 (solid squares)and pH 6 (solid triangles) were recognized by the MPV.A4 monoclonalantibody.

In summary, both MPV.10.34.d capsid backbones refolded from IBs(Example 1) and soluble MPV.10.34.d capsid backbones (Example 4) areboth recognized by the MPV.A4 conformational monoclonal antibody.

Example 6 IRC Formation: MPV.10.34.d Capsid Backbone Conjugation

To functionalize the MPV.10.34.d capsid backbones such that they areeffective in recruiting preexisting immune system to attack cancer cellsin the subject, the MPV.10.34.d capsid backbones were conjugated tovarious peptide epitopes including ovalbumin peptide SIINFEKL (OVA, SEQID NO: 95), HPV16 E7 protein (SEQ ID NO: 96), and CMV peptide pp65 (SEQID NO: 129) to form IRCs.

Design of Peptides: The peptides are epitopes having a general length ofabout 8 to 10 amino acids that are preceded upstream by a proteaserecognition site. (See, FIG. 11). The following experiments incorporatean exemplary protease recognition site, the furin protease cleavagesequence R X R/K R (SEQ ID NO:89) which is designed to be locatedupstream of the epitope peptide. In addition, the epitope peptide ischemically modified at the N-terminus to contain maleimide. Theincorporation of maleimide, a sulfhydryl reactive reagent, to theN-terminus of the peptide antigen allows for conjugation of theprotease/peptide to the reduced sulfhydryl groups, i.e., cysteines, onthe MPV.10.34.d capsid backbones. The end production of this reaction isa conjugated MPV.10.34.d capsid backbone.

To conjugate purified MPV.10.34.d capsid backbones of about >95% purity,the MPV.10.34.d were further dialyzed in conjugation reaction buffer (50mM NaPO₄, pH 6.5, 500 mM NaCl, 2 mM EDTA, and 0.05% Tween® 80),exchanging the buffer three times (3±1 hours, 3±1 hours, and overnight16±3 hours, at 2° C. to 8° C.). The next day, the MPV.10.34.d wasadjusted to a final concentration of at least 0.6 μg/μL. The MPV.10.34.dwere then treated with a mild reducing agent,tris(2-carboxyethyl)phosphine (TCEP), for 1 hour without shaking at roomtemperature (21° C.) at a TCEP:MPV.10.34.d ratio of 10:1. Subsequently,the peptide was then added to the reaction at a molar ratio of X10 theamount of MPV.10.34.d. The reaction was shaken at room temperature (21°C.), 200 rpm, for 1 hour. Following conjugation, to remove excess freepeptide, contents from the reaction were subjected to 10 rounds ofAmicon spin filtration (molecular cut-off 100 kDa) at 1000 rcf for 10mins each round. Following this purification step, samples were analyzedby SDS-PAGE stained with Coomassie Brilliant Blue R-250 dye (Bio-Rad,Hercules, Calif., US) to determine percent conjugation (4-20% CRITERION™TGX Stain-Free™ Precast Gels, 10 Well Comb, 30 μL, 1.0 mm, Bio-Rad,Hercules, Calif., US). As seen in FIG. 12, the upper band at about 50kDa was determined to correspond to IRC. (See, FIG. 12, lane 4). Thelower bands represent the MPV.10.34.d lacking epitope peptide. Theseband identities were further confirmed via the conjugated control. (See,FIG. 12, lane 3).

Importantly, the conjugation of MPV.10.34.d yielded a conjugationefficiency of about 50% as determined by densitometry. (See, FIG. 12,lane 4). Percent conjugation was calculated by densitometry. The gelimages were scanned into the computer using manufacturer-recommendedsoftware. Subsequently, the total density of the upper band and lowerband was calculated by drawing a box or freehand shape around each band.The total density of these regions corresponds to the total amount ofprotein in each band. Next, only the upper lane was measured in the sameway. Background density of a suitably matched area on each gel wascollected and subtracted from the band signals. The background-correcteddensity of the upper protein band was then divided by the entirebackground-corrected density of the region of interest and thenmultiplied by 100 to obtain the percent conjugation.

Example 7 Conjugation Efficiency of MPV.10.34.d and HPV16 CapsidBackbones

Conjugation of HPV L1 particles using the same conjugation reactionsteps as described in Example 6 has been previously described. (See, forinstance, WO 2018/237115 and WO 2020/139978). Conjugation experimentswere conducted on HPV16 capsid backbones and MPV.10.34.d capsidbackbones in the manner described in Example 6. The peptide epitopeconjugated to the capsid backbones was the HLA-A*0201 restricted epitopeNLVPMVATV (NLV, SEQ ID NO: 138) from the HLA-A2 supertype derived fromthe CMV pp65 antigen.

As seen in FIG. 13, under the same conjugation reaction conditions of10:1 (TCEP:L1) and 10:1 (Peptide:L1) ratios, the MPV.10.34.d capsidbackbones exhibited a higher conjugation percentage (50%, FIG. 13, lane5) as compared to HPV16 (approximately 20%, FIG. 13, lane 3). Percentconjugation was determined by densitometry as described above. Controlsamples included unconjugated HPV16 (FIG. 13, lane 2) and unconjugatedMPV.10.34.d (FIG. 13, lane 4).

The percent peptide conjugation is believed to depend on at least twofactors: (1) the ratio of reducing agent to L1 protein, and (2) theamount of free peptide added to the conjugation reaction. For theresults shown in FIG. 13, it was determined that a ratio of reducingagent to L1 of 10:1 and peptide to L1 ratio of 10:1 results in at least50% conjugation for MPV.10.34.d capsid backbone (FIG. 13, lane 5) and atleast 20% for WT HPV16 T=7 capsid backbone (FIG. 13, lane 3).

To further assess impact of reducing agent concentration, theconjugation reaction was performed at 10:1 reducing agent to L1 protein(FIG. 14, lane 3 and lane 10 for HPV16 IRC or MPV.10.34.d IRC,respectively), a 100-fold ratio (FIG. 14, lane 4 and lane 11, for HPV16IRC or MPV.10.34.d IRC, respectively), and a 1000-fold ratio (FIG. 14,lane 5 and lane 12, for HPV16 IRC or MPV.10.34.d IRC, respectively).Surprisingly, a ratio of reducing agent to L1 of 100:1 and 1000:1yielded a lower percent conjugation, and in some instances even nodetectable conjugation, as compared with the standard 10:1 reducingagent to L1 protein ratio. Without wishing to be bound by any specifictheory, it is possible that excess reducing agent reacts with thepeptides to form an -ylene by-product with approximately the same rateas conjugation of peptides to L1 surface thiols. Such a phenomenon couldresult in depletion of peptides available for conjugation to L1.

The relative stability of IRC was also assessed. Following conjugation,the IRC samples were filtered with an Amicon 10 kDa filter spin columnto remove excess free peptide. Following filtration, the proteinconcentration of the samples were checked to ensure no protein was lostduring filtration. (See, FIG. 14, lanes 6 to 8 and lanes 13 to 15, forHPV16 IRC or MPV.10.34.d IRC, respectively). The samples were thenanalyzed by TEM as described in Example 2. FIGS. 15A and 15B areexemplary TEM micrographs at 150,000× and 200,000× magnification,respectively, showing the breakdown of the HPV16 IRC after Amiconpurification. In contrast, MPV.10.34.d IRC did not exhibit anybreakdown, as shown in FIGS. 15C and 15D at 180,000× and 200,000×magnification, respectively.

Without wishing to be bound by any specific theory, it is postulatedthat perhaps the additional stability of the MPV.10.34.d IRC may be dueto the inherent structural stability of the capsid backbone itself,being held together by hydrophobic bonds, as compared to T=7 particlesthat are held together instead by disulfide bonds. Indeed, the reducingstep during the conjugation reaction may in fact destabilize the T=7structures held together by disulfide bonds via reduction of thenecessary thiol groups. As a consequence, the MPV.10.34.d capsidbackbone may be comparatively more stable after being treated with up toa ratio of 100:1 or 1000:1 of TCEP to L1.

As a result of these findings it was concluded that MPV.10.34.d capsidbackbone would serve well as a stable conjugation platform to recruitpreexisting immune system components in a subject for the purpose oftreating cancer in subjects.

Although there is no improvement to conjugation (50% as seen bydensitometry on SDS-PAGE gel) of MPV.10.34.d capsid backbone at reducingagent ratios above 1:100, reducing agent ratios higher than 1:10 butlower than 1:100 were investigated to determine whether such rangesmight increase conjugation efficiency. Thus, the conjugation reactionswere repeated as previously described with varying amounts of reducingagent ratios under 1:100, specifically ratios of 5:1, 10:1, and 25:1.Peptide to MPV.10.34.d ratios (5:1, 10:1, and 25:1) were also evaluated.

As seen in FIG. 16, a dose-dependent peptide conjugation on MPV.10.34.dcapsid backbone using 5:1, 10:1 and 25:1 peptide:L1 ratio when thereducing agent: L1 protein ratio is between 5:1 (lanes 1, 4, and 7) wasobserved. No peptide dose dependence was seen with reducing agent: L1protein ratio at 10:1 (lanes 2, 5, and 8) and 20:1 (lanes 3, 6, and 9).Lane 6 is a reference point in which 10:1 reducing agent to L1 ratio and10:1 peptide to L1 ratio were used, conditions under which anapproximately 50% level of peptide conjugation is routinely observed asdetermined by densitometry. The lane labelled “CEX-FB035” is a controlcontaining only MPV.10.34.d capsid backbones.

It was determined that a 5:1 ratio of reducing agent to L1 along with a10:1 ratio of peptide to L1 yielded a conjugation efficiency above 50%.(See, FIG. 16, lanes 4 and 7). Conjugation conditions with ratios of1:1, 2.5:1, and 5:1 reducing agent to L1 protein were also tested. Theresults obtained at these ratios are reported in FIG. 17. It wasdetermined that lowering the amount of the reducing agent from 5:1 to1:1 did not improve conjugation efficiency, regardless of the amount ofpeptide included in the reactions. In FIG. 17, lane R is a referencepoint sample in which a peptide to L1 ratio of 10:1 was included as wellas reducing agent to L1 ratio of 10:1, which typically yields anapproximately 50% level of peptide conjugation. The lane labelled“CEX-FB035” is a control containing only MPV.10.34.d capsid backbones.In summary, it was determined that a 5:1 reducing agent to L1 ratio anda 10:1 peptide to L1 ratio achieves conjugation rates above 50%.

Example 8 Binding of IRC to Tumor Cells via HSPG

To assess whether IRC bind to tumor cells, an in vitro cell bindingassay was conducted. Specifically, both MPV.10.34.d capsid backbones(unconjugated) as well as different conjugated IRC (human CMV pp65,murine E7, and murine OVA peptide) were examined.

Briefly, 2×10⁵ MC38 cells (murine colon adenocarcinoma, # ENH204-FPKerafast, Inc., Boston Mass.) or pgsA-745 cells (Chinese hamster ovarycell mutant deficient in xylosyltransferase(UDP-D-xylose:serine-1,3-D-xylosyltransferase, ATCC CRL-2242) in whichheparin sulfate proteoglycan (HSPG) expression is knocked out, wereseeded overnight. The next day, the cells were treated with human CMVpp65, murine HPV16 E7, and murine OVA peptide, as well as theMPV.10.34.d capsid backbone for one hour at 37° C. Cells were thenwashed twice with 2 to 3 mL of a fluorescence activated cell sorting(FACS) buffer (1% bovine serum albumin in PBS) and then stained with 1mL of rabbit anti-musPsV serum antibodies for 30 minutes at 4° C.Following this, samples were washed once with 3 mL FACS buffer andstained with 0.5 mL of donkey anti-rabbit IgG-PE antibody (Biolegend,San Diego, Calif.) for 30 min at 4° C. in the dark. Finally, sampleswere washed once more with 3 mL FACS buffer and then resuspended in 250mL of FACS buffer before being analyzed by a CytoFLEX flow cytometer(Beckman Coulter Life Sciences, Brea, Calif., US).

As shown in FIG. 18A and FIG. 18B, all of the constructs,MPV.10.34.d-CMV pp65 IRC (solid line), MPV.10.34.d-E7 IRC (thick solidline), MPV.10.34.d-OVA IRC (thick dashed line), and MPV.10.34.d capsidbackbones (dashed-line), exhibited specificity for tumor cells asevidences by the peak shifts to the right. The positive control in theseexperiments was MPV capsid backbone (wild type, dotted line). Thenegative control included no IRC or L1 (long-dashed line).

These experiments further show that the IRC exhibited HSPG-specificbinding since no binding of MPV.10.34.d capsid backbones was observed inthe cell line lacking HSPG expression (pgsA-745 cells, indicated by noshift in the peaks in FIG. 17B). In summary, these results show thatbinding specificity of MPV.10.34.d capsid backbones for tumor cells isHSPG specific, and importantly, conjugation of epitope peptides toMPV.10.34.d capsid backbone does not reduce or otherwise negativelyimpact binding of MPV.10.34.d IRC to tumor cells in vitro.

Example 9

Loading of Peptide onto Tumor Cells by MPV.10.34.d IRC

The MPV.10.34.d IRC are designed such that upon entering the tumormicroenvironment, the peptide will be cleaved from the IRC, therebyreleasing the peptide in the near vicinity of a tumor cell surface. Thecleavage event occurs, in some embodiments, upon contact with atumor-specific protease, i.e., a protease present, in some embodimentsat relatively higher concentrations than elsewhere in the subject'ssystem, on or nearby a tumor cell. This cleavage event then is designedto result in the loading, or binding, of the peptide by MHC moleculesexpressed on the surface of tumor cells. The following experiments aredesigned to test this mode of operation and whether the designed IRCoperate in the manner expected.

For this purpose, an MHC class I molecule loading assay was developedthat directly detects peptide loading from IRC onto MHC class Imolecules expressed on the surface of tumor cells. This assay involvesthe use of an antibody that specifically recognizes an OVA peptide(SIINFEKL, SEQ ID NO:95)—MHC class I alloantigen H-2K^(b) moleculecomplex but not free peptide, empty MHC class 1 molecules, or peptidesconjugated to the IRC. (See, Zhang et al., Proc. Nat'l. Acad. Sci. USA,89:8403-84-7, 1992).

In this experiment, the OVA conjugated MPV.10.34.d IRC from Example 6were examined side-by-side with OVA conjugated HPV16 IRC at equivalentmolarity based on concentration of conjugated peptide. Briefly, 0.1 to0.2×10⁶ MC38 tumor cells (C57BL6 murine colon adenocarcinoma-derivedcells, # ENH204-FP, Kerafast, Inc., Boston Mass.) were incubated withthe IRC for one hour at 37° C. A positive control including just freepeptide and a negative control including no peptide or IRC were alsotested. Cells were then washed twice with 2 to 3 mL FACS buffer and thenstained with PE-conjugated-mouse anti-mouse MHC I bounded with OVA(SIINFEKL, SEQ ID NO:95) monoclonal antibody (Biolegend, San Diego,Calif.) for 30 minutes at 4° C. Following this, samples were washed oncewith 3 mL FACS buffer then the cells were resuspended in 250 μL of FACSbuffer before being analyzed by a CytoFLEX flow cytometer (BeckmanCoulter Life Sciences, Brea, Calif., US).

Results of these assays are provided in FIG. 19. The results show thatOVA-conjugated MPV.10.34.d IRC (1.4 μg/mL, thick solid line) andOVA-conjugated HPV16 IRC (2.5 μg/mL, solid line) demonstrated loading ofepitopes on the surface of MC38 murine tumor cells, with theOVA-conjugated MPV.10.34.d IRC out-performing the OVA-conjugated HPV16IRC. (See, FIG. 18, negative control—long-dashed line, positivecontrol—thin-dashed line). These results suggest that the MPV.10.34.dIRC is superior to the HPV16 IRC because a smaller amount of MPV.10.34.dIRC achieved the same, or better, “loading” potential of a larger amountof HPV16 IRC.

As OVA is a model antigen utilized for murine MHCs, this experiment wasrepeated substituting the CMV pp65 peptide for the OVA peptide. TheHLA-A*0201 restricted epitope NLVPMVATV (NLV, SEQ ID NO: 138) from theCMV pp65 was used for these studies and the pp65-conjugated MPV.10.34.dIRC were produced as described in Example 6. As there was nocommercially available monoclonal antibody that recognizes an MHC classI—NLV complex, a soluble T-cell receptor antibody (2S16) was employedthat recognizes this HLA-A2 complex. (See, Wagner et al., J. Biol.Chem., 295(15):5790-5804, 2019). The IRC constructs were analyzed in thesame manner as above except that the cell lines HCT116 (human colorectalcarcinoma cell line, HCT 116, ATCC, CCL-247) and MCF7 (human breastcancer cell line, MCF7, ATCC, HTB-22) were utilized in this study. Thesecell lines are HLA-A*0201 restricted and thus are able to present theHLA-A*0201 restricted epitope NLVPMVATV (NLV, SEQ ID NO: 138) from theCMV pp65 peptide.

Consistent with the OVA MHC class I loading results, loading of the NLVpeptide onto human tumor cells was observed. Results are presented inFIG. 19A (HCT116 cells) and FIG. 19B (MCF7 cells). In FIG. 20A and FIG.20B, an unrelated hepatitis B peptide was used as a negative control(thin dashed line, 10 μg/mL), unconjugated MPV.10.34.d capsid backboneswere used as a further negative control (thin solid line, 100 μg/mL),CMV conjugated MPV.10.34.d IRC is represented as a thick solid line (100μg/mL, at about 1.7 μg/mL of hCMV peptide conjugated), hCMV free peptideis represented as a thick dashed line (1 μg/mL).

FIGS. 19 and 20 show that incubation of the MPV.10.34.d IRC in vitrowith the indicated cell lines leads to release of the peptide andbinding to the tumor cell surface MHC Class 1 molecules. To furtherdemonstrate that the mechanism of the IRC first involves the MPV.10.34.dIRC binding to the tumor cell followed by furin cleavage, competitiveinhibition experiments were performed that either inhibited tumor cellbinding or furin cleavage to show that ablation of either step resultsin an absence of peptide loading onto the tumor cells. These studieswere performed with the OVA-conjugated MPV.10.34.d IRC and conductedunder the same conditions as the binding assays described above.

To block tumor binding, soluble heparin (Sigma Aldrich, St. Louis, Mo.)at 1 mg/mL, 5 mg/mL, or 10 mg/mL was incubated with 2.5 μg/mL ofOVA-conjugated MPV.10.34.d IRC for 1 hour at 37° C., 5% CO₂ in thepresence of 2×10⁵ MC38 cells in a FACs tube. The final volume of thecells with the sample was 200 μL. A positive control sample was includedwhich contained no soluble heparin as well as a negative control thatcontained no IRC or heparin. Cells were then washed twice with 2 to 3 mLFACS buffer and then stained with PE-conjugated-mouse anti-mouse MHC Ibound to the OVA peptide monoclonal antibody (this monoclonal antibodyis able to specifically detect OVA peptide, SIINFEKL, SEQ ID NO: 95, incomplex with MHC-I K^(b)) for 30 minutes at 4° C. Following this,samples were washed once with 3 mL FACS buffer, then the cells wereresuspended in 250 μL of FACS buffer before being analyzed by a CytoFLEXflow cytometer (Beckman Coulter Life Sciences, Brea, Calif., US). Asseen in FIGS. 21A, 21B, and 21C, no OVA peptide loading was observed inthe negative control (thin black line in FIGS. 21A, 21B, and 21C). NoOVA peptide loading was observed in samples including 10 mg/ml (dashedline, FIG. 21A), 5 mg/ml (dashed line, FIG. 20B), or 1 mg/ml (dashedline, FIG. 21C) soluble heparin (these curves overlapped with thenegative control data). Loading of OVA peptide was only detected in thesamples containing OVA-conjugated MPV.10.34.d IRC with no heparin (thickblack line to the right, FIGS. 21A, 21B, and 21C). These results showthat OVA-conjugated MPV.10.34.d IRC is HSPG-specific.

To show that loading of peptide from IRC onto tumor cells is dependenton protease cleavage of the epitope peptide form the MPV.10.34.d IRC,the experiments of Example 10 were repeated in the presence of a furininhibitor, furin inhibitor I—Calbiochem, decanoyl-RVKR-CMKa, peptidylchloromethylketone. (Millipore-Sigma, St. Louis, Mo.). This furininhibitor binds irreversibly to the catalytic site of furin, blockingall furin protease activity.

Briefly, 2×10⁵ MC38 cells (murine colon adenocarcinoma, # ENH204-FP,Kerafast, Inc., Boston Mass.) were seeded in a FACs tube and thenincubated with either 0.5 μM, 5 μM, or 50 μM furin inhibitor dissolvedin DMSO at total final sample volume of 200 μL. Control samplescontaining no inhibitor were prepared the same way with the same volumeequivalent of DMSO. The samples were incubated for fifteen minutes in atissue culture incubator at 37° C., 5% CO₂. Then, 2.5 μg/mL ofOVA-conjugated MPV.10.34.d IRC was added to all samples and the sampleswere incubated in a tissue culture incubator at 37° C., 5% CO₂. Sampleswere then washed twice with 2 to 3 mL FACS buffer and then stained withPE-conjugated-mouse anti-mouse MHC I bounded with OVA (SIINFEKL, SEQ IDNO:95) monoclonal antibody (Biolegend, Cat #141604, San Diego, Calif.)for 30 minutes at 4° C. Following this, samples were washed once with 3mL FACS buffer then the cells were resuspended in 250 μL of FACS bufferbefore being analyzed by a CytoFLEX flow cytometer. (Beckman CoulterLife Sciences, Brea, Calif., US).

As seen in FIGS. 22A, 22B, and 22C, OVA-conjugated MPV.10.34.d IRCloaded OVA peptide onto tumor cells in the samples that had no inhibitoradded (thin black line, FIGS. 22A, 22B, and 22C), and samples treatedonly with DMSO and no furin inhibitor (dashed black line, FIGS. 22A,22B, and 22C). In contrast, no OVA peptide loading was observed in thenegative control (thin grey line in FIGS. 22A, 22B, and 22C, the controlcurves overlapped with the experimental data) as well as samples treatedwith furin inhibitor at 50 μM (arrow pointing to dark thin line, FIG.22A), 5 μM (arrow pointing to dark thin line, FIG. 22B), and 0.5 μM(arrow pointing to dark thin line, FIG. 22C). Therefore, inhibition offurin cleavage of the epitope peptide from the IRC prevented binding ofOVA to the MHC molecules of the target cancer cell, thereby confirmingthe mechanism of action of the IRC. This mechanism is further confirmedby, and consistent with, the results shown in FIGS. 18 to 22.

Example 10

In Vitro Cytotoxic Killing Assays with MPV.10.34.d IRC

Since it was shown in Example 9, that in vitro MPV.10.34.d IRC were ableto deposit peptide epitopes onto murine and human MHC Class I moleculesand that this mechanism was dependent on furin activity, additionalexperiments were designed to determine whether labelling these cancercells would trigger activation and redirection of cellular immune systemcomponents against target tumor cells. Upon activation and redirection,the goal is delivery of a cytotoxic signal to the tumor cells and tumorcell death. For this purpose, three different in vitro cytotoxicT-cell-dependent tumor cell killing assays were designed involving theco-culture of tumor cells and viral antigen-specific CD8+ T cells in thepresence or absence of MPV.10.34.d IRC. The three CD8 T-cells weretested, including: (1) murine OVA-specific preclinical CD8+ T cells, (2)murine HPV16 E7-specific CD8+ T cells, and (3) humanHLA-A*0201-restricted CMV-specific T cells (Astarte Biologicals, cat#1049-4367JY19). (See, Example 12, for (3)).

Murine B16 (melanoma/skin) (B16-F10 (ATCC® CRL-6475™), and murine ID8(ovarian) tumor cells (Hung et al., Gene Ther., 14(12):921-020, 2007)overexpressing luciferase gene (B16-luc and ID8-luc) were grown inculture. Under normal circumstances, murine tumor cell lines B16 and ID8will not be killed by murine OVA-specific CD8+ T cells since these celllines do not express the murine OVA (SIINFIKEL, SEQ ID NO: 95) antigen.

Approximately 0.01×10⁶ B16-luciferase mutant (B16-luc) or 0.005×10⁶ID8-luciferase mutant (ID8-luc) tumor cells were seeded in 100 μL perwell on a 96-well assay plate overnight. The cells were then treatedwith 100 μL of 2.5 μg/mL of MPV.10.34.d capsid backbones, OVA-conjugatedMPV.10.34.d IRC, OVA-conjugated HPV16 IRC, and positive controlcontaining 1 μg/mL of free OVA peptide (SIINFIKEL, SEQ ID NO: 95), forone hour at 37° C. in a final volume of 200 μL per well. Cells notreceiving any antigen were included as negative control (No Ag). Thecells were then washed twice with 200 μL of Roswell Park MemorialInstitute (RPMI) media and co-incubated with OVA-specific CD8+ T-cells(Jackson, stock no. 003831) at an effector (CD8+ T-cell) to target cell(tumor cell) ratio (“E:T Ratio”) of 10:1 (B16-luc) or 20:1 (ID8-luc) for16 hours in a final volume of 200 μL per well in a cell incubator at 37°C., 5% CO₂. An E:T ratio of 10:1 means that for every 1 tumor cell, tenCD8+OT-1 T cells will be co-incubated with the tumor cell. Theseco-incubated cells were then washed with 200 μL of PBS and lysed with 35μL of 1× cell lysis buffer (Promega, Madison, Wis., US) for 15 to 20minutes before adding 50 μL of luciferase assay substrate and detectedon a Promega GloMax Explorer Microplate Reader (Promega, Madison, Wis.,US).

The number of viable tumor cells after co-incubation with T cells weremeasured by quantification of concentration of luciferase released fromlysed cells. This acts as a surrogate marker for cell viability sincethe target cells were incubated under conditions in which theyover-express luciferase. Reduced luciferase activity indicated more celldeath suggesting greater immune redirection and hence greatercytotoxicity.

As shown in FIG. 23A and FIG. 23B, the OVA-conjugated MPV.10.34.d IRC,OVA-conjugated HPV16 IRC, and the peptide positive control showed muchhigher tumor cell cytotoxicity (>70%) than the negative control samplesin both B16-luc (FIG. 23A) and ID8-luc (FIG. 23B) tumor killing assays.At the same concentration (2.5 μg/mL), OVA-conjugated MPV.10.34.d IRCalso showed similar high cytotoxicity on tumor cells as theOVA-conjugated HPV16 IRC.

Similar to the above experiment, a second experiment was performed inlike manner, except with the substitution of the E7 peptide (RAHYNIVTF,SEQ ID NO:96) for the OVA peptide. The same tumor cells and samples wereinvestigated in this experiment using the same protocol.

As shown in FIG. 24A and FIG. 24B, the E7-conjugated MPV.10.34.d IRC,E7-conjugated HPV16 IRC, and the positive control showed much highertumor cell cytotoxicity (>70%) than the negative control groups in bothB16-luc (FIG. 24A) and ID8-luc (FIG. 24B) tumor killing assays. At thesame concentration (2.5 μg/mL), OVA-conjugated MPV.10.34.d IRC alsoshowed similar high cytotoxicity on tumor cells as the OVA-conjugatedHPV16 IRC.

Example 11 MPV.10.34.d IRC Effectiveness in Human Assays

While the in vitro functional test results of the above experiments werepromising, the next desired step in the analysis was to perform similarexperiments in human-based assays. To this end, the response of mockhuman cellular immune system components to tumor cells exposed toMPV.10.34.d IRC was examined in vitro. Human CMV (HCMV) was selected forthis study since human CMV is highly prevalent (infecting 50-90% of thehuman population) and mostly asymptomatic in healthy individuals. (See,Longmate et al., Immunogenetics, 52(3-4):165-73, 2001; Pardieck et al.,F1000Res, 7, 2018; and van den Berg et al., Med. Microbiol. Immunol.,208(3-4):365-373, 2019). Importantly, HCMV establishes a life-longpersistent infection that requires long-lived cellular immunity toprevent disease. Hence, it is rational to hypothesize that a complexadaptive cell-mediated anti-viral immunity developed over many years tostrongly control a viral infection in an aging person can be repurposedand harnessed to treat cancer.

In these experiments, CD8+ T cell responses to CMV peptides were testedin three different human tumor cell lines, including HCT116, OVCAR3, andMCF7. All three of these human tumor cell lines are HLA-A*0201 positive.

In vitro cytotoxicity assays. HTC112, human colon cancer cells, MCF7,human breast cancer cells, and OVCAR3, human ovarian cancer cells (allfrom ATCC, Manassas, Va., US) were seeded overnight at 0.01 to 0.2×10⁶per well per 100 μL per 96 well plate. The next day (about 20 to 22 hrslater), each cell line was incubated for one hour at 37° C. under thefollowing conditions: (1) CMV peptide at a final concentration of 1μg/mL (positive control), (2) MPV.10.34.d at a final concentration of2.5 μg/mL (negative control), (3) CMV-conjugated MPV.10.34.d IRC at afinal concentration of 2.5 μg/mL, (4) CMV-conjugated HPV16 IRC at afinal concentration of 2.5 μg/mL, and (5) no antigen (negative control).After 1 hour, the cells were washed vigorously with 200 μL of media forthree times to remove non-specific binding. Human patient donor CMV Tcells (ASTARTE Biologics, Seattle, Wash., US) were added at the E:T(effector cell:target cell) ratio of 10:1 and incubated in a tissueculture incubator for 24 hrs at 37C, 5% CO₂. The total final volume ofeach sample after co-culture was 200 μL. Cell viability was measuredafter co-culturing. Cell viability was measured with CELLTITER-GLO®(Promega, Madison, Wis., US). This assay provides aluciferase-expressing chemical probe that detects and binds to ATP, amarker of cell viability. The amount of ATP generated from tumor cellswas quantified according to manufacturer protocols. In these assays,reduced luciferase activity indicates cell death and suggests greaterimmune redirection and greater cytotoxicity.

The results are provided in FIG. 25. CMV-conjugated MPV.10.34.d IRC(“VERI-101” in FIGS. 25A, 25B, and 25C) was equally effective asCMV-conjugated HPV16 IRC (“CMV AIR-VLP” in FIGS. 25A, 25B, and 25C) inredirecting human healthy donor CMV pp65-specific CD8+ T-cells (AstarteBiologics, Inc., Bothell, Wash., US) to kill immortalized HLA.A2positive human colon cancer cells (HCT116), human ovarian cancer cells(OVCAR3), and human breast cancer cells (MCF7). The control samples (“NoAg” or “VERI-000” in FIGS. 25A, 25B, and 25C) showed no background tumorkilling. Together, these data demonstrate that MPV.10.34.d IRC redirectsmouse and human immune responses against tumor cells in vitro.

Example 12 Sequential Mechanism of MPV.10.34.d IRC Binding and PeptideCleavage

Example 9 demonstrates that MPV.10.34.d IRC binding must occur prior tofurin-dependent cleavage of the peptide and peptide loading onto targettumor cells. A dose-response curve using different concentrations ofOVA-conjugated MPV.10.34.d IRC to detect binding and loading in separateassays was generated. These assays were performed as described inExamples 7 and 8. Based on the geometric MFIs from both assays, acorrelation analysis was conducted.

The results shown in FIG. 26 indicate that there is a highlystatistically significant correlation between the number ofOVA-conjugated MPV.10.34.d IRC binding to tumor cells with the level ofthe OVA peptide/K^(b) complex on the tumor cells (Spearman r=0.92,P=0.0003; Pearson r=0.98, p<0.001). This statistical analysis furtherdemonstrates the requirement for the sequential steps of OVA-conjugatedMPV.10.34.d IRC to first bind or contact the tumor cell, followed byfurin-dependent cleavage of the peptide from the IRC, and MHC loading ofthe peptide.

Example 13 Sequential Mechanism of MPV.10.34.d IRC Binding and TumorCell Death

Example 9 shows that inhibition of OVA-conjugated MPV.10.34.d IRCbinding results in inhibition of furin-dependent cleavage of the peptidefrom the IRC and OVA peptide loading onto tumor cell surfaces. Tofurther show that inhibition of this binding step also inhibitsredirection of CD8+ T-cells and tumor cell death, cytotoxicity studiesconducted as in Example 10 were performed in the presence and absence ofsoluble heparin, a competitor of HSPG binding.

A range of OVA-conjugated MPV.10.34.d IRC concentrations (0.156m/mL to0.625 μg/mL) as well as E:T ratios (1:4.5, 1:9 and 1:18) wereinvestigated in the presence and absence of 10 mg/mL of soluble heparinin the assays described in Example 10. This concentration of solubleheparin was previously shown to cause complete inhibition ofOVA-conjugated MPV.10.34.d IRC binding, as well as inhibition of peptideloading onto tumor cells. In these assays, 15,000 TC-1 cellsoverexpressing luciferase were first seeded in a flat-bottom 96-wellplate overnight in a cell culture incubator at 37° C., 5% CO₂. The nextday, cells were washed 3 times with PBS before being incubated withAIM-V media (serum free) with 2% BSA for 1.5 hours in a cell cultureincubator at 37° C., 5% CO₂. In parallel, OVA-conjugated MPV.10.34.d IRCwas diluted in the same AIM-V media+2% BSA into 0.625, 0.3125, 0.156μg/mL. (See, FIGS. 27A, 27B, and 27C). Each sample was incubated with(thick dash lines) or without (solid line) 10 mg/mL of soluble heparinfor 1 hour at 2° C. to 8° C. MPV.10.34.d capsid backbones (thin dashline, “ViP” only) was included as a negative control. After 1 hour, thesamples were added to the TC-1 cells and co-incubated for a further 30minutes in a cell culture incubator at 37° C., 5% CO₂. Following this,treated cells were washed 3 times with just AIM-V media before beingincubated with OT-1 T-cells at an effector to target (E:T) ratio of18:1, 9:1, or 4.5:1. This co-culture was then incubated for a further 3hours at 37° C., 5% CO₂. After 3 hours, target cells were analyzed forcytotoxicity using the Promega Luciferase Assay system as per themanufacturer's protocol (Promega, Madison, Wis., US). Cytotoxicity wasdetermined by detection of loss of luciferase signal which is used as asurrogate marker of cell viability in this assay. All studies wereperformed in triplicate.

Results are shown in FIG. 27. The presence of soluble heparin exhibitedno OVA-conjugated MPV.10.34.d IRC-mediated cytotoxicity under allconcentrations and E:T ratio conditions tested. These results furthersubstantiate the sequential nature of the MPV.10.34.d IRC mechanism ofaction.

A correlation analysis was conducted on the binding and cytotoxicityactivities of OVA-conjugated MPV.10.34.d IRC. Briefly, a dose-responsecurve using different concentrations of OVA-conjugated MPV.10.34.d IRCto detect binding and cytotoxicity in separate assays was generated.Cytotoxicity assays were conducted as previously described in Example 10with the following changes: a range of 6.25×10⁻⁵m/mL to 2.5 μg/mL ofOVA-conjugated MPV.10.34.d IRC was tested at 3 different E:T ratios(18:1, 9:1, and 4.5:1).

Under all 3 E:T ratio conditions tested, a dose dependent killing wasobserved with OVA-conjugated MPV.10.34.d IRC concentrations below 0.04μg/mL and higher, whereas concentrations of OVA-conjugated MPV.10.34.dIRC between 0.156 μg/mL to 2.5 μg/mL lead to a maximal level ofcytotoxicity. Binding assays were conducted according to the protocolsdescribed in Example 7 with the following changes: a concentration rangeof 6.24×10⁻⁴ to 2.5 μg/mL of OVA-conjugated MPV.10.34.d IRC wasinvestigated.

Results show that a dose-dependent binding was observed and that thelimit of binding detection was reached at 2.5×10⁻⁴m/mL. Both assays wererepeated twice (with at least 3 replicates). The mean values ofgeometric mean fluorescent intensity (MFI) was reported from the twoexperiments and is summarized in FIG. 28.

Based on the MFIs from both assays (FIG. 28), a graphical andcorrelation analysis was conducted using Spearman correlational analysis(FIG. 29). Briefly, the mean of the percentages of two independentOVA-conjugated MPV.10.34.d IRC cytotoxicity assays performed on twodifferent days and the mean of MFIs of OVA-conjugated MPV.10.34.d IRCbinding experiments from two different days were calculated (FIG. 28)and plotted (FIG. 29). Spearman correlational analysis was performed onthese results reveals a significant relationship (r=0.83-0.9) betweenthese two variables at all three E:T ratios. These results show thatthere is a highly statistically significant correlation (r value between0.83 to 0.9, depending on E:T ratio) between the OVA-conjugatedMPV.10.34.d IRC binding to tumor cells and the level of cytotoxicitythat followed.

Example 14 GARDASIL®9-Generated Antibodies do Not Inhibit MPV.10.34.dIRC Effects

Vaccination with GARDASIL®9 results in long term (>10 years) ofsustained HPV L1 capsid-specific antibodies that are able to prevent HPVinfection and subsequently, prevent HPV-associated cervical cancers.Although GARDASIL®9 has been reported to be only effective against ninetypes of HPVs, some cross-neutralization against other types ofpapillomavirus capsids may be expected. As MPV.10.34.d IRC is derivedfrom murine papillomavirus capsids, it was desirable to determinewhether vaccine sera elicited from GARDASIL®9 vaccination could inhibitMPV.10.34.d IRC tumor cell killing.

GARDASIL®9 sera was generated as follows: New Zealand white rabbits(n=10) were administered three intra-muscular vaccinations of a humandose of GARDASIL®9 (270 μg of VLPs per dose). Rabbits were vaccinated atmonths 0, 1, and 2. After two weeks post final vaccination, rabbits werebled to obtain the GARDASIL®9 sera. 100 μL aliquots of sera from eachrabbit were pooled. As a control, GARDASIL®9 sera were also tested forneutralizing activity against HPV types 6, 11, 16, 18, 31, 45, 52, and58 and results showed no neutralization activity (data not shown).

OVA-conjugated MPV.10.34.d IRCs were tested with GARDASIL®9 sera usingthe protocol described in Examples 11 and 13. Briefly, 15,000 TC-1 cellsoverexpressing luciferase were first seeded in a flat-bottom 96-wellplate overnight in a cell culture incubator at 37° C., 5% CO₂. The nextday, cells were washed 3 times with PBS before being incubated withAIM-V media (serum free)+2% BSA for 1.5 hours in a cell cultureincubator at 37° C., 5% CO₂. In parallel, OVA-conjugated MPV.10.34.d IRCwas diluted in the same AIM-V media+2% BSA into 0.625 μg/mL, 0.3125μg/mL, and 0.156 μg/mL, and each sample was incubated with a 1:200dilution of GARDASIL®9 serum (thick-dashed lines) or without (solidline) for 1 hour at 2° C. to 8° C. MPV.10.34.d alone (thin dashed line)was also included as a negative control. (See, FIGS. 30A, 30B, and 30C).After 1 hour, the samples were added to the TC-1 cells and co-incubatedfor 30 minutes in a cell culture incubator at 37° C., 5% CO₂. Followingthis, treated cells were washed 3 times with just AIM-V media beforebeing incubated with OT-1 T-cells at an effector to target (E: T) ratioof 18:1, 9:1, or 4.5:1. This co-culture was then incubated for 3 hoursat 37° C., 5% CO₂. After 3 hours, target cells were analyzed forcytotoxicity using the Promega Luciferase Assay system as per themanufacturer's protocol (Promega, Madison, Wis., US). Cytotoxicity wasdetermined by quantitation of the loss of luciferase signal which isused as a surrogate marker of cell viability. All studies were performedin triplicate.

No inhibition of cytotoxicity was observed in the presence of GARDASIL®9sera. The results in FIG. 30 suggests that MPV.10.34.d IRC would not benegatively impacted in subjects who might possess preexisting HPVvaccine-generated antibodies.

Example 15 Anti-MPV.10.34.d IRC Antibodies do Not Inhibit Tumor CellCytotoxicity

Since MPV.10.34.d IRC sequences are based on MPV L1 capsids, it wasdesirable to test whether antibodies generated against wild type MPV orMPV.10.34.d IRC affect the mechanism of action of MPV.10.34.d IRCsagainst tumors.

Antibodies against wild type MPV were generated as follows: New Zealandwhite rabbits (n=3) were administered three intra-muscular vaccinationsof 50 μg of wild type mouse papillomavirus particles per dose. Rabbitswere vaccinated at months 0, 1, and 2. After two weeks post finalvaccination, rabbits were bled to obtain the anti-MPV sera. Antibodiesagainst MPV.10.34.d IRC were obtained as follows: naïve 6 to 8 week oldC57/BL6 mice (n=10) were injected systemically with two doses of 150 μgof E7-conjugated MPV.10.34.d over a period of 48 hours. After 48 hours,the mice were bled to obtain the anti-MPV.10.34.d IRC sera.

Specificity of anti-MPV.10.34.d IRC sera and anti-MPV sera to bothMPV.10.34.d capsid backbone and MPV.10.34.d IRC was examined by ELISA.The assays were performed as described in Example 5 with the followingdifferences: serum samples were tested at a 1:100 dilution factor andtwo-fold serial dilutions. Goat anti-mouse IgG-HRP secondary antibodywas used in the ELISA (1:7000). Binding of anti-MPV.10.34.d IRC sera(FIG. 31) and anti-MPV serum (data not shown) to both MPV.10.34.d capsidbackbone (FIG. 31A) and MPV.10.34.d IRC (FIG. 31B) was observed.

To determine whether binding of either antibody serum to MPV.10.34.dIRCs would affect the subsequent tumor cytotoxicity, binding andcytotoxicity assays were conducted with OVA-conjugated MPV.10.34.d IRCin the presence or absence of either sera. Cytotoxicity assays wereconducted as in Example 14 in the presence of anti-MPV serum (FIGS. 32A,32B, and 32C) or anti-MPV.10.34.d IRC serum (FIGS. 32D, 32E, and 32F).Results reveal that no difference in cytotoxicity when OVA-conjugatedMPV.10.34.d IRC was pre-incubated with either anti-MPV sera (FIGS. 32A,32B, and 32C) or anti-MPV.10.34.d IRC sera (FIGS. 32D, 32E, and 32F) forall concentrations and E:T ratios tested.

The inability of antibodies to MPV.10.34.d IRC or MPV to inhibitcytotoxicity despite these antibodies showing specificity for binding toboth MPV.10.34.d capsid backbones and MPV.10.34.d IRCs via ELISA wasfurther investigated by conducting binding assays in the presence ofthese sera.

Samples were pre-incubated with different OVA-conjugated MPV.10.34.d IRCconcentrations (0.0025 μg/mL to 2.5 μg/mL). These samples were tested inthe same binding assays described in Example 5. Briefly, 20,000 TC-1cells overexpressing luciferase were seeded into FACs tubes andincubated in a cell culture incubator at 37° C., 5% CO₂, until needed.In parallel, OVA-conjugated MPV.10.34.d IRC was diluted in the sameAIM-V media +2% BSA into a range of concentrations 0.0025 to 2.5 μg/mLand each sample was incubated with 1:200 dilution of either anti-MPVserum (A) or anti-MPV.10.34.d IRC serum (B) for 1 hour at 2° C. to 8° C.After 1 hour, the samples were added to the TC-1 cells seeded in theFACs tubes and co-incubated for 1 hour in a cell culture incubator at37° C., 5% CO₂. Samples were then washed twice with FACS buffer (DBPS,pH 7, 0.1% BSA). The samples were then stained with AF647-conjugateddonkey anti-rabbit Ig antibody or PE-conjugated goat anti-mouse IgGantibody for 30 minutes in the dark. Samples were then washed with FACSbuffer (DBPS, pH 7, 0.1% BSA). After this, samples were resuspending in250 μL of FACs buffer and binding was detected by flow cytometry atdifferent concentrations of OVA-conjugated MPV.10.34.d IRC pre-treatedwith MPV sera (FIG. 33A) or anti-MPV.10.34.d IRC sera (FIG. 33B). Allstudies were performed in triplicate.

Results of these tests reveals that incubation of samples with anti-MPVrabbit IgG serum (FIG. 33A) or anti-MPV.10.34.d IRC mouse IgG serum(FIG. 33B) are still able to bind to target tumor cells. In summary, theresults from Example 15 (FIGS. 31, 32, and 33) collectively show thatantibodies specific for MPV.10.34.d IRCs elicited in vivo bindspecifically to the MPV.10.34.d IRCs. However, these antibodies do notblock MPV.10.34.d IRCs from binding to tumor cells (FIG. 33) and thus,also do not inhibit the overall cytotoxic mechanism of the MPV.10.34.dIRCs (FIG. 32). In contrast, these same antibodies were able to inhibitMPV infection as seen in the pseudo-virus neutralization assays (datanot shown). As MPV infection requires cell internalization, one possibleexplanation is that these anti-MPV.10.34.d IRC serum antibodies blockcell internalization of MPV.10.34.d IRC but do not inhibit target cellbinding of MPV.10.34.d IRC. Since the mechanism of MPV.10.34.d IRCs isextracellular, it is possible that this is why they are not affected bythese antibodies.

Example 16 Peptide Loading on Cells Deficient in MHC Class 1Intracellular Pathway Components

Example 9 demonstrates loading of peptide from IRCs onto the tumor cellsurface. It was desirable to determine whether the released peptidesfrom the IRC are bound by the tumor cell and then phagocytosed to beprocessed through the MHC Class 1 antigen presentation pathway. To testthis possibility, peptide loading assays were performed with a cell line(RMA-S cells) that is genetically deficient (TAP-deficient) inintracellular MHC Class 1 processing proteins. If peptide loading ontothe tumor cell surface still occurs in this context, it must be throughextracellular mechanisms.

A range of OVA-conjugated MPV.10.34.d IRC concentrations (0.625m/mL to10 μg/mL) was tested for their ability to load RMA-S cells with peptide.These binding assays were conducted as previously described. Briefly,RMA-S cells at 2×10⁶ cells/mL were resuspended into a single cellsuspension and 100 μL (0.2×10⁶ of RMA-S cells) was dispensed into FACStubes. Varying amounts of OVA-conjugated MPV.10.34.d IRC was added(0.625 μg/mL to 10 μg/mL) to the samples and the samples were thenincubated at 37° C., 5% CO₂ for one hour. Afterwards, the cells werewashed twice in 2 mL of FACS buffer (PBS, pH7.0, 1% BSA). The cells werestained with 1 μL of PE-conjugated anti-SIINFEKL (SEQ ID NO:95)/Kbantibody. The cells were washed twice in 2 mL of FACS buffer (PBS,pH7.0, 1% BSA). The samples were then resuspended in about 250 μL ofFACS buffer and peptide binding was analyzed via MFI.

The data reveal that increasing levels of OVA peptide-MHC complex inthese cells as higher concentrations of OVA-conjugated MPV.10.34.d IRCswere added. These data appear to establish that the MPV.10.34.d IRCslabel target tumor cells by an extracellular mechanism.

What is claimed is:
 1. A composition, comprising: a plurality of virusproteins, wherein each of said plurality of virus proteins comprises amutated amino acid sequence of a Papillomaviridae L1 protein; one ormore peptides each comprising one or more epitopes from one or morepathogens other than a Papillomaviridae antigenic peptide; wherein themutated amino acid sequence of the Papillomaviridae L1 protein comprisesat least the following mutations with respect to the wild type L1protein sequence: (a) a deletion of at least five amino acid residuesfrom an amino-terminus, and (b) a deletion of at least ten amino acidresidues from the helix four region, wherein the one or more peptidesare attached to the plurality of virus proteins, and wherein saidplurality of virus proteins spontaneously assemble to form anicosahedron or dodecahedron capsid having a triangulation number T equalto 1 that binds to proteoglycan expressed on tumor cells.
 2. Thecomposition of claim 1, wherein the amino acid sequence of each of theplurality of mutant Papillomaviridae L1 proteins further comprises: (c)a deletion of at least thirty amino acid residues from acarboxy-terminus.
 3. The composition of claim 1, wherein the one or morepeptides are conjugated to the plurality of mutant Papillomaviridae L1proteins.
 4. The composition of claim 3, wherein the one or morepeptides are conjugated to the mutant Papillomaviridae L1 proteins via acysteine, lysine, or arginine residue of the mutant Papillomaviridae L1protein.
 5. The composition of claim 3, wherein the one or more peptidesare conjugated to the mutant Papillomaviridae L1 proteins viadisulphide, maleimide, or amide bond between the mutant PapillomaviridaeL1 protein and a residue of the peptide.
 6. The composition of claim 1,wherein at least 25% of the plurality of mutant Papillomaviridae L1proteins are conjugated to at least one of the one or more peptides. 7.The composition of claim 1, wherein at least 25 to 85% (w/w) of theplurality of mutant Papillomaviridae L1 proteins are conjugated to atleast one of the one or more peptides.
 8. The composition of claim 1,wherein each of the one or more peptides further comprise one or moreprotease cleavage sequences.
 9. The composition of claim 8, wherein theone or more protease cleavage sequences comprise a furin cleavagesequence, a matrix metalloprotease cleavage sequence, or a disintegrinand metalloprotease (ADAM) cleavage sequence.
 10. The composition ofclaim 1, wherein the one or more epitopes are viral, bacterial,parasitic, or fungal epitopes.
 11. The composition of claim 10, wherein:the viral epitopes are one or more of coronavirus, vaccinia, Varicellazoster, Herpes zoster, rubella, hepatitis, influenza, measles, mumps,poliovirus, variola, rabies, dengue, Ebola, West Nile, yellow fever, orzika epitopes; the bacterial epitopes are one or more of Bordetellapertussis, Clostridium tetani, Chlamydia trachomatis, Corynebacteriumdiphtheriae, Hemophilus influenza, Neisseria meningitidis,Streptococcus, Vibrio cholera, Mycobacterium tuberculosis, BacillusCalmette-Guérin, Salmonella, Escherichia coli, Legionella pneumophila,Rickettsia, Treponema pallidum pallidum, Bacillus anthracis, Clostridiumbotulinum, or Yersinia epitopes; or the parasitic epitopes are one ormore of Entamoeba histolytica, Toxoplasma gondii, Trichinella,Trichomonas, Trypanosoma, or Plasmodium epitopes.
 12. The composition ofclaim 1, wherein at least one of the one or more epitopes is a childhoodvaccine antigenic epitope.
 13. The composition of claim 1, wherein theone or more peptides comprises at least two epitopes from one or morepathogens other than a Papillomaviridae antigenic peptides.
 14. Thecomposition of claim 1, wherein the proteoglycan expressed on tumorcells is heparin sulfate proteoglycan (HSPG), perlecan, hyalectanversican, glypican-3, small leucine-rich proteoglycans (SLRP), and/orbiglycan.
 15. The composition of claim 1, wherein the plurality ofmutant Papillomaviridae L1 proteins do not form a T=7 capsid backbone.16. The composition of claim 1, wherein said plurality of mutantPapillomaviridae L1 proteins are mouse mutant Papillomaviridae L1proteins.
 17. The composition of claim 1, wherein an amino acid sequenceof each of said plurality of mutant Papillomaviridae L1 proteins is SEQID NO:134, and is encoded by nucleic acid sequence SEQ ID NO:135 or 136.18. A method of treating, reducing the occurrence of, inhibiting theprogression and/or metastasis of, a cancer in a subject in need thereof,which comprises administering to the subject a pharmaceuticallyeffective amount of a composition comprising: a plurality of virusproteins, wherein each of said plurality of virus proteins comprises amutated amino acid sequence of a Papillomaviridae L1 protein; one ormore peptides each comprising one or more epitopes from one or morepathogens other than a Papillomaviridae antigenic peptide; wherein themutated amino acid sequence of the Papillomaviridae L1 protein comprisesat least the following mutations with respect to the wild type L1protein sequence: (a) a deletion of at least five amino acid residuesfrom an amino-terminus, and (b) a deletion of at least ten amino acidresidues from the helix four region, wherein the one or more peptidesare attached to the plurality of virus proteins, and wherein saidplurality of virus proteins spontaneously assemble to form anicosahedron or dodecahedron capsid having a triangulation number T equalto 1 that binds to proteoglycan expressed on tumor cells.
 19. The methodof claim 18, further comprising: obtaining from the subject a tumortissue sample; and identifying in the tumor tissue a sequence of one ormore MHC molecules expressed by one or more tumor cells in the tumortissue sample.
 20. The method of claim 18, wherein the subject waspreviously infected or vaccinated against a pathogen, and wherein theone or more epitopes is an antigenic epitope of the pathogen.
 21. Themethod of claim 18, wherein the one or more epitopes are capable ofcomplexing with one or more MHC molecules expressed by a tumor cell in atumor tissue sample obtained from the subject.
 22. A process forproducing the composition of claim 1, which comprises: (a) transforminga prokaryotic cell with an expression vector encoding the L1 protein;(b) culturing the transformed prokaryotic cell under conditions thatpromote expression of the L1 protein; (c) lysing the transformedprokaryotic cells to release expressed L1 protein; (d) separating celldebris from the expressed L1 protein and recovering the L1 protein asinclusion bodies; (e) optionally washing the L1 protein inclusionbodies; (f) solubilizing the L1 protein inclusion bodies; (g) refoldingthe L1 protein; and (h) forming the icosahedron or dodecahedron capsidhaving a triangulation number T equal to 1 by incubating the refolded L1protein in refolding buffer.
 23. The process of claim 22, furthercomprising: (i) conjugating in a conjugation buffer the one or morepeptides to the assembled L1 protein by incubating the assembled L1protein under reducing conditions in the presence of one or morepeptides.
 24. The process of claim 22, wherein the refolding buffercomprises a denaturant, a reducing agent, 250 to 500 mM of a salt, anon-ionic surfactant, a metal chelating agent, and a buffer of pH 7.5 to8.5.
 25. The process of claim 24, wherein the salt is present from 250mM to 500 mM.
 26. The process of claim 24, wherein the molar ratio ofthe one or more peptides to the L1 protein is at least 1:5.
 27. Theprocess of claim 22, further comprising removing denaturant from therefolding buffer but maintaining reducing agent when forming theicosahedron or dodecahedron capsid having a triangulation number T equalto 1.